Novel 3-substituted 5-amino-6h-thiazolo[4,5-d]pyrimidine-2, 7-dione compounds for the treatment and prophylaxis of virus infection

ABSTRACT

wherein R1, R2 and R3 are as described herein, and their prodrugs or pharmaceutically acceptable salt, enantiomer or diastereomer thereof, and compositions including the compounds and methods of using the compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/207,338, filed Jul. 11, 2016 which is a continuation of U.S.application Ser. No. 14/958,619, filed Dec. 3, 2015 which claims thebenefit under 35 U.S.C § 119(a) to International ApplicationPCT/CN2015/086987, filed Aug. 14, 2015, and claims the benefit under 35U.S.C § 119(a) to International Application PCT/CN2014/093224, filedDec. 8, 2014. The entire contents of these applications are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel 3-substituted5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione compounds, that haveToll-like receptor agonism activity and their prodrugs thereof, as wellas their manufacture, pharmaceutical compositions containing them andtheir potential use as medicaments.

The present invention relates to compounds of formula (I) and (Ia),

and their prodrugs, formula (II) and (IIa),

wherein R¹ to R⁶ are described below, or pharmaceutically acceptablesalt, enantiomer or diastereomer thereof.

Toll-like receptors (TLRs) detect a wide range of conservedpathogen-associated molecular patterns (PAMPs). They play an importantrole of sensing invading pathogens and subsequent initiation of innateimmune responses. There are 10 known members of the TLR family in human,which are type I transmembrane proteins featuring an extracellularleucine-rich domain and a cytoplasmic tail that contains a conservedToll/interleukin (IL)-1 receptor (TIR) domain. Within this family, TLR3,TLR7 TLR8, and TLR9 are located within endosomes. TLR7 can be activatedby binding to a specific small molecule ligand (i.e., TLR7 agonist) orits native ligand (i.e., single-stranded RNA, ssRNA). Following bindingof ssRNA to TLR7, the receptor in its dimerized form is believed toundergo a structural change leading to the subsequent recruitment ofadapter proteins at its cytoplasmic domain, including the myeloiddifferentiation primary response gene 88 (MyD88). Following theinitiation of the receptor signalling cascade via the MyD88 pathway,cytoplasmic transcription factors such as interferon regulatory factor 7(IRF-7) and nuclear factor kappa B (NF-κB) are activated. Thesetranscription factors then translocate to the nucleus and initiate thetranscription of various genes, e.g., IFN-α and other antiviral cytokinegenes. TLR7 is predominately expressed on plasmacytoid cells, and alsoon B-cells. Altered responsiveness of immune cells might contribute tothe reduced innate immune responses during chronic viral infections.Agonist-induced activation of TLR7 might therefore represent a novelapproach for the treatment of chronic viral infections. (D. J Connollyand L. A J O'Neill, Current Opinion in Pharmacology 2012, 12:510-518, P.A. Roethle et al, J. Med. Chem. 2013, 56, 7324-7333).

The current therapy of chronic HBV infection is based on two differenttypes of drugs: the traditional antiviral nucleos(t)ide analogues andthe more recent Pegylated IFN-α (PEG-IFN-α). The oral nucleos(t)ideanalogues act by suppressing the HBV replication. This is a life-longcourse of treatment during which drug resistance often occurs. As analternative option, Pegylated IFN-α (PEG-IFN-α) has been used to treatsome chronic infected HBV patients within finite therapy duration.Although it has achieved seroconversion in HBeAg at least in a smallpercentage of HBV patients, the adverse effect makes it poorlytolerable. Notably, functional cure defined as HBsAg seroconversion isvery rare with both current therapies. A new generation therapeuticoption to treat HBV patients for a functional cure is therefore ofurgent need. Treatment with an oral TLR7 agonist represents a promisingsolution to provide greater efficacy with better tolerability. PegylatedIFN-α (PEG-IFN-α) is currently used to treat chronic HBV and is analternative to potentially life-long treatment with antiviralnucleos(t)ide analogues. In a subset of chronic HBV patients, PEG-IFN-αtherapy can induce sustained immunologic control of the virus followinga finite duration of therapy. However, the percentage of HBV patientsthat achieve seroconversion with interferon therapy is low (up to 27%for HBeAg-positive patients) and the treatment is typically poorlytolerated. Furthermore, functional cure (defined as HBsAg loss andseroconversion) is also very infrequent with both PEG-IFN-α andnucleos(t)ide treatment. Given these limitations, there is an urgentneed for improved therapeutic options to treat and induce a functionalcure for chronic HBV. Treatment with an oral, small-molecule TLR7agonist is a promising approach that has the potential to providegreater efficacy and tolerability (T. Asselah et al, Clin Liver Dis2007, 11, 839-849).

In fact, several identified TLR7 agonists have been considered fortherapeutic purposes. So far Imiquimod (ALDARA™) is a U.S. FDA approvedTLR7 agonist drug for topical use to treat skin lesions by humanpapillomavirus. The TLR7/8 dual agonist resiquimod (R-848) and the TLR7agonist 852A have been evaluated for treating human genital herpes andchemotherapy-refractory metastatic melanoma, respectively. ANA773 is anoral pro-drug TLR7 agonist, developed for the treatment of patients withchronic hepatitis C virus (HCV) infection and chronic hepatitis Binfection. GS-9620 is an orally available TLR7 agonist. A phase Ib studydemonstrated that treatment with GS-9620 was safe, well tolerated andresulted in dose-dependent ISG15 mRNA induction in patients with chronichepatitis B (E. J. Gane et al, Annu Meet Am Assoc Study Liver Dis(November 1-5, Washington, D.C.) 2013, Abst 946). Therefore there ishigh unmet clinical need for developing potent and safe TLR7 agonists asnew HBV treatment to offer more therapeutic solutions or replaceexisting partly effective treatment.

SUMMARY OF THE INVENTION

The present invention provides a series of novel 3-substituted5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione compounds, that haveToll-like receptor agonism activity and their prodrugs. The inventionalso provides the bio-activity of such compounds to induce SEAP levelincrease by activating Toll-like receptors, such as TLR7 receptor, themetabolic conversion of prodrugs to parent compounds in the presence ofhuman hepatocytes, and the therapeutic or prophylactic use of suchcompounds and their pharmaceutical compositions comprising thesecompounds and their prodrugs to treat or prevent infectious disease likeHBV or HCV. The present invention also provides compounds with superioractivity. In addition, the compounds of formula (I) and (Ia) also showedgood solubility and PK profiles.

The present invention relates to novel compounds of formula (I) and(Ia),

wherein

R¹ is hydroxy, C₁₋₆alkyl, haloC₁₋₆alkyl, C₁₋₆alkylcarbonyl-O—,C₁₋₆alkyl-S—, azido, cyano, C₂₋₆alkenyl, C₁₋₆alkylsulfonyl-NH—,(C₁₋₆alkyl)₂N—, C₁₋₆alkylcarbonyl-NH— or heterocyclic amino;

R² is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkyl, C₃₋₇cycloalkyl,C₂₋₆alkynyl, C₂₋₆alkenyl, benzyl and thiophenyl;

R³ is hydrogen or C₁₋₆alkylcarbonyl;

or pharmaceutically acceptable salt, enantiomer or diastereomer thereof;with the proviso that5-amino-7-hydroxy-3-[3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;[2-(5-amino-7-hydroxy-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]acetate;[4-acetoxy-5-(5-amino-7-hydroxy-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylacetate and their diastereomers are excluded.

The present invention also relates to the prodrugs of formula (II) and(IIa),

wherein

R⁴ is hydroxy, C₁₋₆alkyl, haloC₁₋₆alkyl, C₁₋₆alkylcarbonyl-O—,C₁₋₆alkyl-S—, azido, cyano, C₂₋₆alkenyl, C₁₋₆alkylsulfonyl-NH—,(C₁₋₆alkyl)₂N—, C₁₋₆alkylcarbonyl-NH— or heterocyclic amino;

R⁵ is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkyl, C₃₋₇cycloalkyl,C₂₋₆alkynyl, C₂₋₆alkenyl, benzyl and thiophenyl;

R⁶ is hydrogen or C₁₋₆alkylcarbonyl;

or pharmaceutically acceptable salt, enantiomer or diastereomer thereof;with the proviso that5-amino-3-[3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;[2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]acetate;[4-acetoxy-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylacetate and their diastereomers are excluded.

The invention also relates to their manufacture, medicaments based on acompound in accordance with the invention and their production as wellas the use of compounds of formula (I) or (Ia) or their prodrugs,formula (II) or (IIa), thereof as TLR7 agonist. Accordingly, thecompounds of formula (I) and (Ia) or their prodrugs of formula (II) and(IIa) are useful for the treatment or prophylaxis of HBV and/or HCVinfection with Toll-like receptors agonism.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Furthermore, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the various terms used to describe the invention.

Definitions

As used herein, the term “C₁₋₆alkyl” denotes a saturated, linear orbranched chain alkyl group containing 1 to 6, particularly 1 to 4 carbonatoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl and the like. Particular “C₁₋₆alkyl” groups aremethyl, ethyl and n-propyl.

The term “halogen” and “halo” are used interchangeably herein and denotefluoro, chloro, bromo, or iodo.

The term “haloC₁₋₆alkyl” refers to an alkyl group wherein at least oneof the hydrogen atoms of the alkyl group has been replaced by same ordifferent halogen atoms, particularly fluoro atoms. Examples ofhaloC₁₋₆alkyl include monofluoro-, difluoro- or trifluoro-methyl, -ethylor -propyl, for example 3,3,3-trifluoropropyl, 2-fluoroethyl,2,2,2-trifluoroethyl, fluoromethyl, difluoromethyl and trifluoromethyl.

The term “heterocyclic” ring denotes a monovalent saturated or partlyunsaturated mono or bicyclic ring system of 3 to 10 ring atoms,comprising 1 to 5 ring heteroatoms selected from N, O and S, theremaining ring atoms being carbon. In particular embodiments,heterocyclic ring is a monovalent saturated monocyclic ring system of 4to 7 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N,O and S, the remaining ring atoms being carbon. Examples for monocyclicsaturated heterocyclic ring are aziridinyl, oxiranyl, azetidinyl,oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl,pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl,azepanyl, diazepanyl, homopiperazinyl, and oxazepanyl. Examples forbicyclic saturated heterocyclic ring are 8-aza-bicyclo[3.2.1]octyl,quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl,9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, and3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples for partly unsaturatedheterocyclic ring are dihydrofuryl, imidazolinyl, dihydro-oxazolyl,tetrahydro-pyridinyl, and dihydropyranyl.

The term “heterocyclic amino” denotes an amino group with the nitrogenatom on the heterocyclic ring.

The term “C₂₋₆alkenyl” denotes an unsaturated, linear or branched chainalkenyl group containing 2 to 6, particularly 2 to 4 carbon atoms, forexample vinyl, propenyl, allyl, butenyl and the like. Particular“C₂₋₆alkenyl” groups are allyl and vinyl.

The term “C₂₋₆alkynyl” denotes an unsaturated, linear or branched chainalkynyl group containing 2 to 6, particularly 2 to 4 carbon atoms, forexample ethynyl, 1-propynyl, propargyl, butynyl and the like. Particular“C₂₋₆alkynyl” groups are ethynyl and 1-propynyl.

The term “C₃₋₇cycloalkyl”, alone or in combination, refers to asaturated carbon ring containing from 3 to 7 carbon atoms, particularlyfrom 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like. Particular“C₃₋₇cycloalkyl” group is cyclopropyl.

The term “carbonyl” alone or in combination refers to the group —C(O)—.

The term “C₁₋₆alkylcarbonyl” refers to a group C₁₋₆alkyl-C(O)—, whereinthe “C₁₋₆alkyl” is as defined above. Particular “C₁₋₆alkylcarbonyl”group is acetyl.

The term “enantiomer” denotes two stereoisomers of a compound which arenon-superimposable mirror images of one another.

The term “diastereomer” denotes a stereoisomer with two or more centersof chirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, and polyamine resins.

Compounds of the general formula (I) or (Ia) and their prodrugs whichcontain one or several chiral centers can either be present asracemates, diastereomeric mixtures, or optically active single isomers.The racemates can be separated according to known methods into theenantiomers. Particularly, diastereomeric salts which can be separatedby crystallization are formed from the racemic mixtures by reaction withan optically active acid such as e.g. D- or L-tartaric acid, mandelicacid, malic acid, lactic acid or camphorsulfonic acid.

The compounds of the invention may exhibit the phenomenon oftautomerism. While the formula drawings cannot expressly depict allpossible tautomeric forms, it is to be understood they are intended torepresent any tautomeric form of the depicted compound and are not to belimited merely to a specific compound form depicted by the formuladrawings. For example, it is understood for formula (III) thatregardless of whether or not the substituents are shown in their enol ortheir keto form, they represent the same compound (as shown in theexample below).

R_(x) refers to any feasible substituent.

Some of the inventive compounds may exist as single stereoisomers (i.e.,essentially free of other stereoisomers), racemates, and/or mixtures ofenantiomers and/or diastereomers. All such single stereoisomers,racemates and mixtures thereof are intended to be within the scope ofthe present invention. Preferably, the inventive compounds that areoptically active are used in optically pure form. As generallyunderstood by those skilled in the art, an optically pure compoundhaving one chiral center (i.e., one asymmetric carbon atom) is one thatconsists essentially of one of the two possible enantiomers (i.e., isenantiomerically pure), and an optically pure compound having more thanone chiral center is one that is both diastereomerically pure andenantiomerically pure. Preferably, the compounds of the presentinvention are used in a form that is at least 90% optically pure, thatis, a form that contains at least 90% of a single isomer (80%enantiomeric excess (“e.e.”) or diastereomeric excess (“d.e.”)), morepreferably at least 95% (90% e.e. or d.e.), even more preferably atleast 97.5% (95% e.e. or d.e.), and most preferably at least 99% (98%e.e. or d.e.). Additionally, compounds of formula (I) and (Ia) and theirprodrugs, formula (II) and (IIa), and other compounds of the inventionare intended to cover solvated as well as unsolvated forms of theidentified structures. For example, formula (I) or (Ia) includescompounds of the indicated structure in both hydrated and non-hydratedforms. Other examples of solvates include the structures in combinationwith isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,or ethanolamine.

The term “prodrug” denotes a form or derivative of a compound which ismetabolized in vivo, e.g., by biological fluids or enzymes by a subjectafter administration, into a pharmacologically active form of thecompound in order to produce the desired pharmacological effect.Prodrugs are described e.g. in “The Organic Chemistry of Drug Design andDrug Action”, by Richard B. Silverman, Academic Press, San Diego, 2004,Chapter 8 Prodrugs and Drug Delivery Systems, pp. 497-558.

“A pharmaceutically active metabolite” is intended to mean apharmacologically active product produced through metabolism in the bodyof a specified compound or salt thereof. After entry into the body, mostdrugs are substrates for chemical reactions that may change theirphysical properties and biologic effects. These metabolic conversions,which usually affect the polarity of the compounds of the invention,alter the way in which drugs are distributed in and excreted from thebody. However, in some cases, metabolism of a drug is required fortherapeutic effect.

The term “therapeutically effective amount” denotes an amount of acompound or molecule of the present invention that, when administered toa subject, (i) treats or prevents the particular disease, condition ordisorder, (ii) attenuates, ameliorates or eliminates one or moresymptoms of the particular disease, condition, or disorder, or (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition or disorder described herein. The therapeuticallyeffective amount will vary depending on the compound, the disease statebeing treated, the severity of the disease treated, the age and relativehealth of the subject, the route and form of administration, thejudgement of the attending medical or veterinary practitioner, and otherfactors.

The term “pharmaceutical composition” denotes a mixture or solutioncomprising a therapeutically effective amount of an activepharmaceutical ingredient together with pharmaceutically acceptableexcipients to be administered to a mammal, e.g., a human in needthereof.

TLR7 Agonist and Prodrug

The present invention relates to a compound of formula (I),

wherein

R¹ is hydroxy, C₁₋₆alkyl, haloC₁₋₆alkyl, C₁₋₆alkylcarbonyl-O—,C₁₋₆alkyl-S—, azido, cyano, C₂₋₆alkenyl, C₁₋₆alkylsulfonyl-NH—,(C₁₋₆alkyl)₂N—, C₁₋₆alkylcarbonyl-NH— or heterocyclic amino;

R² is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkyl, C₃₋₇cycloalkyl,C₂₋₆alkynyl, C₂₋₆alkenyl, benzyl and thiophenyl;

R³ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof;-   with the proviso that    5-amino-7-hydroxy-3-[3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;    [2-(5-amino-7-hydroxy-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]    acetate;    [4-acetoxy-5-(5-amino-7-hydroxy-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methyl    acetate and their diastereomers are excluded.

Further embodiment of present invention is (ii) a compound of formula(I), wherein

R¹ is hydroxy, methyl, propyl, fluoroisopropyl, acetyloxy,methylsulfanyl, azido, cyano, allyl, 2-methylallyl, methylsulfonylamino,dimethyl amino, acetylamino, pyrrolidinyl, morpholinyl or piperidinyl;

R² is hydrogen, methyl, ethyl, propyl, butyl, cyclopropyl, cyclopentyl,vinyl, allyl, benzyl, ethynyl, 1-propynyl, methoxymethyl or thiophenyl;

R³ is hydrogen, acetyl or isobutyryl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is (iii) a compound of formula(Ia),

wherein

R¹ is hydroxy, C₁₋₆alkyl, haloC₁₋₆alkyl, C₁₋₆alkylcarbonyl-O—,C₁₋₆alkyl-S—, azido, cyano, C₂₋₆alkenyl, C₁₋₆alkylsulfonyl-NH—,(C₁₋₆alkyl)₂N—, C₁₋₆alkylcarbonyl-NH— or heterocyclic amino;

R² is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkyl, C₃₋₇cycloalkyl,C₂₋₆alkynyl, C₂₋₆alkenyl, benzyl or thiophenyl;

R³ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof;-   with the proviso that    5-amino-7-hydroxy-3-[3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;    [2-(5-amino-7-hydroxy-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]    acetate;    [4-acetoxy-5-(5-amino-7-hydroxy-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methyl    acetate and their diastereomers are excluded.

Further embodiment of present invention is (iv) a compound of formula(Ia), wherein

R¹ is hydroxy, methyl, propyl, fluoroisopropyl, acetyloxy,methylsulfanyl, azido, cyano, allyl, 2-methylallyl, methylsulfonylamino,dimethyl amino, acetylamino, pyrrolidinyl, morpholinyl or piperidinyl;

R² is hydrogen, methyl, ethyl, propyl, butyl, cyclopropyl, cyclopentyl,vinyl, allyl, benzyl, ethynyl, 1-propynyl, methoxymethyl or thiophenyl;

R³ is hydrogen, acetyl or isobutyryl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is (v) a compound of formula (I)or (Ia), wherein

R¹ is hydroxy, C₁₋₆alkyl, C₁₋₆alkylcarbonyl-O—, C₁₋₆alkyl-S—, azido orC₂₋₆alkenyl;

R² is C₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkyl, C₃₋₇cycloalkyl, C₂₋₆alkynyl,C₂₋₆alkenyl, benzyl and thiophenyl;

R³ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (vi) a compound of formula(I) or (Ia), wherein

R¹ is hydroxy, methyl, propyl, acetyloxy, methylsulfanyl, azido orallyl;

R² is methyl, ethyl, propyl, butyl, cyclopropyl, cyclopentyl, vinyl,allyl, benzyl, ethynyl, 1-propynyl, methoxymethyl or thiophenyl;

R³ is hydrogen, acetyl or isobutyryl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (vii) a compound of formula(I) or (Ia), wherein R¹ is hydroxy, C₁₋₆alkyl, C₁₋₆alkyl-S—, azido orC₂₋₆alkenyl.

A further embodiment of present invention is (viii) a compound offormula (I) or (Ia), wherein R¹ is hydroxy, methyl, n-propyl,methylsulfanyl, azido or allyl.

A further embodiment of present invention is (ix) a compound of formula(I) or (Ia), wherein R² is C₁₋₆alkyl, C₃₋₇cycloalkyl, C₂₋₆alkynyl orC₂₋₆alkenyl.

A further embodiment of present invention is (x) a compound of formula(I) or (Ia), wherein R² is methyl, ethyl, n-propyl, cyclopropyl, vinyl,ethynyl or 1-propynyl.

A further embodiment of present invention is (xi) a compound of formula(I) or (Ia), wherein R³ is hydrogen or C₁₋₆alkylcarbonyl.

A further embodiment of present invention is (xii) a compound of formula(I) or (Ia), wherein R³ is hydrogen or isobutyryl.

Another embodiment of present invention is (xiii) a compound of formula(I) or (Ia), wherein

R¹ is hydroxy, C₁₋₆alkyl, C₁₋₆alkyl-S—, azido or C₂₋₆alkenyl;

R² is C₁₋₆alkyl, C₃₋₇cycloalkyl, C₂₋₆alkynyl or C₂₋₆alkenyl;

R³ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (xiv) a compound of formula(I) or (Ia), wherein

R¹ is hydroxy, methyl, propyl, methylsulfanyl, azido or allyl;

R² is methyl, ethyl, propyl, cyclopropyl, vinyl, ethynyl or 1-propynyl;

R³ is hydrogen or isobutyryl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is (xv) a compound of formula(I) or (Ia), wherein

R¹ is haloC₁₋₆alkyl, C₁₋₆alkyl-S—, cyano, C₂₋₆alkenyl, (C₁₋₆alkyl)₂N—,C₁₋₆alkylcarbonyl-NH— or heterocyclic amino;

R² is hydrogen;

R³ is hydrogen;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (xvi) a compound of formula(I) or (Ia), wherein

R¹ is fluoroisopropyl, methylsulfanyl, cyano, 2-methylallyl, methylsulfonyl amino, dimethylamino, acetyl amino, pyrrolidinyl, morpholinylor piperidinyl;

R² is hydrogen;

R³ is hydrogen;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (xvii) a compound offormula (I) or (Ia), wherein R¹ is C₁₋₆alkyl-S— or heterocyclic amino.

A further embodiment of present invention is (xviii) a compound offormula (I) or (Ia), wherein R¹ is methylsulfanyl or pyrrolidinyl.

Another embodiment of present invention is that (xix) particularcompounds of formula (I) or (Ia) are the following:

-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   [(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(1-hydroxypropyl)tetrahydrofuran-3-yl]    acetate;-   [(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]    acetate;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybut-3-enyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypentyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybutyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[cyclopentyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxy-2-phenyl-ethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxy-3-methyl-butyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[cyclopropyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   [[2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]-cyclopropyl-methyl]    acetate;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyprop-2-ynyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybut-2-ynyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-[hydroxy(2-thienyl)methyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxy-2-methoxy-ethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-(1-hydroxypropyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-azido-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyallyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-((2R,3R,5S)-3-azido-5-((S)-1-hydroxyethyl)tetrahydrofuran-2-yl)thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione;-   3-[(2R,3R,5S)-3-allyl-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-propyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-amino-3-[(2R,3R,5S)-5-[(1R)-1-hydroxypropyl    ]-3-propyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxybut-2-ynyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[(S)-cyclopropyl(hydroxy)methyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxyethyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-pyrrolidin-1-yl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   N-[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]methanesulfonamide;-   N-[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]acetamide;-   5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-morpholino-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-(1-piperidyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-(dimethylamino)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   (2R,3S,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carbonitrile;-   5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-(1-fluoro-1-methyl-ethyl)-5-(hydroxymethyptetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-(2-methylallyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione-   and    [(1S)-1-[(2S,4R,5R)-5-(5-Amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]2-methylpropanoate;-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is that (xx) more particularcompounds of formula (I) or (Ia) are the following:

-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybutyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[cyclopropyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyprop-2-ynyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybut-2-ynyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-(1-hydroxypropyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-azido-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyallyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   3-[(2R,3R,5S)-3-allyl-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-propyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-amino-3-[(2R,3R,5S)-5-[(1R)-1-hydroxypropyl]-3-propyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxybut-2-ynyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[(S)-cyclopropyl(hydroxy)methyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxyethyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-pyrrolidin-1-yl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;-   and    [(1S)-1-[(2S,4R,5R)-5-(5-Amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]2-methylpropanoate;-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is (xxi) a compound of formula(II),

wherein

R⁴ is hydroxy, C₁₋₆alkyl, haloC₁₋₆alkyl, C₁₋₆alkylcarbonyl-O—,C₁₋₆alkyl-S—, azido, cyano, C₂₋₆alkenyl, C₁₋₆alkylsulfonyl-NH—,(C₁₋₆alkyl)₂N—, C₁₋₆alkylcarbonyl-NH— or heterocyclic amino;

R⁵ is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkyl, C₃₋₇cycloalkyl,C₂₋₆alkynyl, C₂₋₆alkenyl, benzyl and thiophenyl;

R⁶ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof;-   with the proviso that    5-amino-3-[3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;    [2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethy)tetrahydrofuran-3-yl]    acetate;    [4-acetoxy-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methyl    acetate and their diastereomers are excluded.

A further embodiment of present invention is (xxii) a compound offormula (II), wherein

R⁴ is hydroxy, methyl, n-propyl, fluoroisopropyl, acetyloxy,methylsulfanyl, azido, cyano, allyl, 2-methylallyl, methylsulfonylamino, dimethylamino, acetylamino, pyrrolidinyl, morpholinyl orpiperidinyl;

R⁵ is hydrogen, methyl, ethyl, propyl, butyl, cyclopropyl, cyclopentyl,vinyl, allyl, benzyl, ethynyl, 1-propynyl, methoxymethyl or thiophenyl;

R⁶ is hydrogen, acetyl or isobutyryl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is (xxiii) a compound of formula(IIa),

wherein

R⁴ is hydroxy, C₁₋₆alkyl, haloC₁₋₆alkyl, C₁₋₆alkylcarbonyl-O—,C₁₋₆alkyl-S—, azido, cyano, C₂₋₆alkenyl, C₁₋₆alkylsulfonyl-NH—,(C₁₋₆alkyl)₂N—, C₁₋₆alkylcarbonyl-NH— or heterocyclic amino;

R⁵ is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkyl, C₃₋₇cycloalkyl,C₂₋₆alkynyl, C₂₋₆alkenyl, benzyl and thiophenyl;

R⁶ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof;-   with the proviso that    5-amino-3-[3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;    [2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]acetate;    [4-acetoxy-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methyl    acetate and their diastereomers are excluded.

A further embodiment of present invention is (xiv) a compound of formula(IIa), wherein

R⁴ is hydroxy, methyl, n-propyl, fluoroisopropyl, acetyloxy,methylsulfanyl, azido, cyano, allyl, 2-methylallyl, methylsulfonylamino, dimethylamino, acetylamino, pyrrolidinyl, morpholinyl orpiperidinyl;

R⁵ is hydrogen, methyl, ethyl, propyl, butyl, cyclopropyl, cyclopentyl,vinyl, allyl, benzyl, ethynyl, 1-propynyl, methoxymethyl or thiophenyl;

R⁶ is hydrogen, acetyl or isobutyryl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is (xxv) a compound of formula(II) or (IIa), wherein

R⁴ is hydroxy, C₁₋₆alkyl, C₁₋₆alkyl-S—, azido or C₂₋₆alkenyl;

R⁵ is C₁₋₆alkyl, C₃₋₇cycloalkyl, C₂₋₆alkynyl or C₂₋₆alkenyl;

R⁶ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (xxvi) a compound offormula (II) or (IIa), wherein

R⁴ is hydroxy, methyl, propyl, methylsulfanyl, azido or allyl;

R⁵ is methyl, ethyl, propyl, cyclopropyl, vinyl, ethynyl or 1-propynyl;

R⁶ is hydrogen or isobutyryl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (xxvii) a compound offormula (II) or (IIa), wherein

R⁴ is hydroxy or C₁₋₆alkylcarbonyl-O—;

R⁵ is C₁₋₆alkyl or C₃₋₇cycloalkyl;

R⁶ is hydrogen or C₁₋₆alkylcarbonyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

A further embodiment of present invention is (xxviii) a compound offormula (II) or (IIa), wherein

R⁴ is hydroxy or acetyloxy;

R⁵ is ethyl or cyclopropyl;

R⁶ is hydrogen or acetyl;

-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Another embodiment of present invention is that (xxix) particularcompounds of formula (II) or (IIa) are the following:

-   5-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;-   [(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(1-hydroxypropyl)tetrahydrofuran-3-yl]acetate;-   1-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl    acetate;-   [(S)-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]-cyclopropyl-methyl]acetate;-   and    5-Amino-3-[(2R,3R,5S)-5-[cyclopropyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;-   or pharmaceutically acceptable salt, enantiomer or diastereomer    thereof.

Synthesis

The compounds of the present invention can be prepared by anyconventional means. Suitable processes for synthesizing these compoundsas well as their starting materials are provided in the schemes belowand in the examples. All substituents, in particular, R¹ to R¹¹ are asdefined above unless otherwise indicated. Furthermore, and unlessexplicitly otherwise stated, all reactions, reaction conditions,abbreviations and symbols have the meanings well known to a person ofordinary skill in organic chemistry.

R^(a) is R² or R⁵; R^(b) is acyl, benzoyl, tert-butyl(dimethyl)silyl ortert-butyl(diphenyl)silyl; R^(c) is hydrogen or hydroxy; R^(d) istrifluoromethylsulfonyl or p-tolylsulfonyl; R^(e) is R^(l) or R⁴.

Treatment of compound V with carbon disulfide and iodomethane in thepresence of an appropriate base, such as NaH, affords methylsulfanylmethanethioate 1a. Deoxylation of methylsulfanylmethanethioate 1a withan appropriate reagent, such as tri-n-butyltin hydride affords 1b.Deprotection of 1b with an appropriate acid, such as acetic acid,affords diol 1c. Oxidation of 1c with oxidant, such as sodiummetaperiodate, gives aldehyde 1d. Treatment of aldehyde 1d with anappropriate nucleophile reagent, such as Grignard reagent, gives alcoholVI. Protection of alcohol VI with an appropriate acid anhydride or acidchloride, such as acetic chloride or benzoyl anhydride, affords esterVII. Treatment of ester VII with acetic acid and acetic anhydride in thepresence of an appropriate acid, such as condensed sulfuric acid,affords ester VIII. Coupling of ester VIII and IX in the presence of anappropriate silicon etherification agent, such asN,O-bis(trimethylsilyl)acetamide, and Lewis acid, such as TMSOTf, givesthe intermediate X. Deprotection of intermediate X with appropriatereagent, such as K₂CO₃ or TBAF, and purification by preparative HPLCaffords desired compounds P1, P2 or P3. Protection of compound P1 withan appropriate acid anhydride or acid chloride, such as acetic chlorideor acetic anhydride, affords compound P3. Oxidation of P2 with anappropriate oxidant, such as Dess-Martin periodinane, affords ketone XI.Reduction of ketone XI with an appropriate reductant, such astert-butoxyaluminum hydride, affords alcohol XII. Treatment of alcoholXII with sulfonic anhydride or sulfonyl chloride affords intermediateXIII Treatment of intermediate XIII with an appropriate nucleophilereagent, such as sodium azide, affords compound XIV. Deprotection of XIVwith an appropriate base, such as K₂CO₃, or an appropriate fluoridereagent, such as TBAF, and purification by preparative HPLC affordscompound P4.

R^(a) is R² or R⁵; R^(c) is hydrogen or hydroxy; R^(e) is R¹ or R⁴.

Acid 28a is converted to acyl chloride 28b with an appropriate reagent,such as oxalyl dichloride. Acyl chloride 28b is treated with anappropriate nucleophile reagent, such as Grignard reagent, to affordketone XV. Reduction of ketone XV with an appropriate reductant, such asL-selectride, affords alcohol XVI. Protection of XVI withtert-butylchlorodiphenylsilane gives intermediate XVII. Alkylation ofXVII with an alkylating reagent, such as iodomethane, in the presence ofan appropriate base, such as lithium diisopropylamide, affordsintermediate XVIII. Deprotection of XVIII with an appropriate reagent,such as TBAF, affords alcohol XIX. Protection of alcohol XIX withbenzoyl chloride affords ester XX. Reduction of ester XX with anappropriate reductant, such as diisobutyl aluminium hydride, affordsalcohol XXI. Esterification of alcohol XXI with acetic chloride oracetic anhydride affords XXII. Coupling of XXII and IX in the presenceof an appropriate silicon etherification agent such asN,O-bis(trimethylsilyl)acetamide and Lewis acid gives XXIII Deprotectionof XXIII with an appropriate base, such as K₂CO₃, and purification bypreparative HPLC affords compound P5.

R^(c) is hydrogen or hydroxy; R^(d) is trifluoromethylsulfonyl orp-tolylsulfonyl; R^(e) is R¹ or R⁴.

Protection of alcohol 25e with benzoyl chloride affords intermediate32a. Deprotection of 32a with an appropriate acid, such ashydrochloride, in the presence of an appropriate solvent, such asmethanol, affords intermediate 32b. Treatment of 32b with with sulfonicanhydride or sulfonyl chloride affords intermediate XXIV. Treatment ofXXIV with a nucleophile reagent, such as sodium azide and amines,affords intermediate XXV. Coupling of XXV and IX in the presence of anappropriate silicon etherification agent such asN,O-bis(trimethylsilyl)acetamide and Lewis acid gives XXVI. Deprotectionof XXVI with an appropriate base, such as K₂CO₃, and purification bypreparative HPLC affords compound XXVII.

R^(a) is R² or R⁵; R^(c) is hydrogen or hydroxy; R^(e) is R¹ or R⁴.

Treatment of XXIX with an appropriate reagent, such as O-phenylchloromethanethioate, in the presence of an appropriate base, such asDMAP, affords intermediate XXX. Treatment of intermediate XXX withorganostannane reagent, such as allyl(tri-n-butyl)stannane, affordsintermediate XXXI. Deprotection of XXXI with an appropriate base, suchas K₂CO₃, and purification by preparative HPLC affords compound P6.

This invention also relates to a process for the preparation of acompound of formula (I), (Ia), (II) or (IIa) comprising the reaction of:

-   (a) the reaction of a compound of formula (X),

with a base or fluoride reagent, wherein R^(a) is R² or R⁵; R^(b) isacyl, benzoyl, tert-butyl(dimethyl)silyl or tert-butyl(diphenyl)silyl;R^(e) is hydrogen or hydroxy;

-   (b) the reaction of a compound of formula (P1),

with an acid anhydride or acid chloride, wherein R^(a) is R² or R⁵;R^(e) is hydrogen or hydroxy;

-   (c) the reaction of a compound of formula (XIV),

with a base or a fluoride reagent, wherein R^(a) is R² or R⁵; R^(b) isacyl, benzoyl, tert-butyl(diphenyl)silyl; R^(e) is hydrogen or hydroxy;R^(d) is trifluoromethylsulfonyl or p-tolylsulfonyl; R^(e) is R¹ or R⁴;

-   (d) the reaction of a compound of formula (XXIII),

with a base, wherein R^(a) is R² or R⁵; R^(c) is hydrogen or hydroxy;R^(e) is R¹ or R⁴;

-   (e) the reaction of a compound of formula (XXVI),

with a base, wherein R^(c) is hydrogen or hydroxy; R^(e) is R¹ or R⁴;

-   (f) the reaction of a compound of formula (XXXI),

with a base, wherein R^(a) is R² or R⁵; R^(c) is hydrogen or hydroxy;R^(e) is R¹ or R⁴; or

wherein R^(a), R^(b), R^(c), R^(d), R^(e), R¹, R², R³, R⁴, R⁵ and R⁶ aredefined above.

In step (a), the base can be for example K₂CO₃, the fluoride reagent canbe for example TBAF.

In step (b), the acid anhydride or acid chloride can be for exampleacetic chloride or acetic anhydride.

In step (c), (d), (e) and (f), the base can be for example K₂CO₃, thefluoride reagent can be for example TBAF.

A compound of formula (I), (Ia), (II) and (IIa) when manufacturedaccording to the above process is also an object of the invention.

Pharmaceutical Compositions and Administration

Another embodiment provides pharmaceutical compositions or medicamentscontaining the compounds of the invention and a therapeutically inertcarrier, diluent or excipient, as well as methods of using the compoundsof the invention to prepare such compositions and medicaments. In oneexample, compounds of formula (I) or (Ia) or their prodrugs may beformulated by mixing at ambient temperature at the appropriate pH, andat the desired degree of purity, with physiologically acceptablecarriers, i.e., carriers that are non-toxic to recipients at the dosagesand concentrations employed into a galenical administration form. The pHof the formulation depends mainly on the particular use and theconcentration of compound, but preferably ranges anywhere from about 3to about 8. In one example, a compound of formula (I) or (Ia) or theirprodrugs are formulated in an acetate buffer, at pH 5. In anotherembodiment, the compounds of formula (I) or (Ia) or their prodrugs aresterile. The compound may be stored, for example, as a solid oramorphous composition, as a lyophilized formulation or as an aqueoussolution.

Compositions are formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners. The “effective amount” of the compoundto be administered will be governed by such considerations, and is theminimum amount necessary to activate TLR7 receptor and lead to produceINF-α and other cytokines, which can be used, but not limited, for thetreatment or prevention of hepatitis B and/or C viral infected patients.

In one example, the pharmaceutically effective amount of the compound ofthe invention administered parenterally per dose will be in the range ofabout 0.1 to 50 mg/kg, alternatively about 0.1 to 30 mg/kg of patientbody weight per day, with the typical initial range of compound usedbeing 0.3 to 15 mg/kg/day. In another embodiment, oral unit dosageforms, such as tablets and capsules, preferably contain from about 20 toabout 1000 mg of the compound of the invention.

The compounds of the invention may be administered by any suitablemeans, including oral, topical (including buccal and sublingual),rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal,intrapulmonary, intradermal, intrathecal and epidural and intranasal,and, if desired for local treatment, intralesional administration.Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration.

The compounds of the present invention may be administered in anyconvenient administrative form, e.g., tablets, powders, capsules,solutions, dispersions, suspensions, syrups, sprays, suppositories,gels, emulsions, patches, etc. Such compositions may contain componentsconventional in pharmaceutical preparations, e.g., diluents, carriers,pH modifiers, sweeteners, bulking agents, and further active agents.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier or excipient. Suitable carriers and excipientsare well known to those skilled in the art and are described in detailin, e.g., Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Formsand Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins,2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice ofPharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe,Raymond C. Handbook of Pharmaceutical Excipients. Chicago,Pharmaceutical Press, 2005. The formulations may also include one ormore buffers, stabilizing agents, surfactants, wetting agents,lubricating agents, emulsifiers, suspending agents, preservatives,antioxidants, opaquing agents, glidants, processing aids, colorants,sweeteners, perfuming agents, flavoring agents, diluents and other knownadditives to provide an elegant presentation of the drug (i.e., acompound of the present invention or pharmaceutical composition thereof)or aid in the manufacturing of the pharmaceutical product (i.e.,medicament).

An example of a suitable oral dosage form is a tablet containing about20 to 1000 mg of the compound of the invention compounded with about 30to 90 mg anhydrous lactose, about 5 to 40 mg sodium croscarmellose,about 5 to 30 mg polyvinylpyrrolidone (PVP) K30, and about 1 to 10 mgmagnesium stearate. The powdered ingredients are first mixed togetherand then mixed with a solution of the PVP. The resulting composition canbe dried, granulated, mixed with the magnesium stearate and compressedto tablet form using conventional equipment. An example of an aerosolformulation can be prepared by dissolving the compound, for example 20to 1000 mg, of the invention in a suitable buffer solution, e.g. aphosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride,if desired. The solution may be filtered, e.g., using a 0.2 micronfilter, to remove impurities and contaminants.

An embodiment, therefore, includes a pharmaceutical compositioncomprising a compound of formula (I) or (Ia) or their prodrugs, formula(II) or (IIa), or pharmaceutically acceptable salts or enantiomers ordiastereomers thereof.

In a further embodiment includes a pharmaceutical composition comprisinga compound of formula (I) or (Ia) or their prodrugs, formula (II) or(IIa), or pharmaceutically acceptable salts or enantiomers ordiastereomers thereof, together with a pharmaceutically acceptablecarrier or excipient.

Another embodiment includes a pharmaceutical composition comprising acompound of formula (I) or (Ia) or their prodrugs, formula (II) or(IIa), or pharmaceutically acceptable salts or enantiomers ordiastereomers thereof for use in the treatment of hepatitis B virusinfection.

Indications and Methods of Treatment

The present invention provides methods for treating or preventing ahepatitis B viral infection and/or hepatitis C viral infection in apatient in need thereof.

The present invention further provides methods for introducing atherapeutically effective amount of a formula (I) or (Ia) compounds ortheir prodrugs, or other compounds of the invention into the bloodstream of a patient in the treatment and/or prevention of hepatitis Band/or C viral infection.

The methods of the present invention are particularly well suited forhuman patients. In particular, the methods and doses of the presentinvention can be useful for, but not limited to, HBV and/or HCV infectedpatients. The methods and doses of the present invention are also usefulfor patients undergoing other antiviral treatments. The preventionmethods of the present invention are particularly useful for patients atrisk of viral infection. These patients include, but are not limited tohealth care workers, e.g., doctors, nurses, hospice care givers;military personnel; teachers; childcare workers; patients traveling to,or living in, foreign locales, in particular third world localesincluding social aid workers, missionaries, and foreign diplomats.Finally, the methods and compositions include the treatment ofrefractory patients or patients resistant to treatment such asresistance to reverse transcriptase inhibitors, protease inhibitors,etc.

Another embodiment includes a method of treating or preventing hepatitisB viral infection and/or hepatitis C viral infection in a mammal in needof such treatment, wherein the method comprises administering to saidmammal a therapeutically effective amount of a compound of formula (I)or (Ia), or enantiomers, diastereomers, prodrugs or pharmaceuticallyacceptable salts thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Example 1-A activates murine TLR7 in a HEK-Blue-mTLR assay. Thecells were incubated with Example 1-A and a positive control GS-9620 atindicated concentrations for 20 hours. The activation of murine TLR7 wasmeasured using a Quanti-Blue assay.

FIG. 2: In vitro cytokine induction by Example 1-A in murine PBMC.Murine PBMC were stimulated with Example 1-A at indicatedconcentrations. Supernatants were collected at 24 hour post treatmentand cytokine levels in supernatants were assessed by a multiplex assay.

FIG. 3: HBV DNA and HBsAg in the AAV-HBV infected mice treated withVehicle, a low dose of Example 6-A at 30 mg/kg, and a high dose ofExample 6-A at 100 mg/kg. The treatment started after the mice wereinfected with AAV-HBV for 29 days. They were given the treatment for 42days, and HBV DNA and HBsAg in mouse serum were measured on theindicated time points by RT-qPCR and HBsAg CLIA respectively. Theresults were presented as mean±SEM. LLQ: lower limit of quantification.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention.

Abbreviations

-   ACN: acetonitrile-   DMAP: 4-dimethylaminopyridine-   CDCl₃: deuterated chloroform-   DCM: dichloromethane-   DMF: dimethyl formamide-   EtOAc: ethyl acetate-   FBS: fetal bovine serum-   HPLC: high performance liquid chromatography-   MS (ESI): mass spectroscopy (electron spray ionization)-   BSA: N, O-bis(trimethylsilyl)acetamide-   NMR: nuclear magnetic resonance obsd. observed-   NaBH₄: sodium borohydride-   TBAF: tetrabutylammonium fluoride-   EC₅₀: The molar concentration of an agonist, which produces 50% of    the maximum possible response for that agonist.-   TEA: triethylamine-   TMSOTf: trimethylsilyl trifluoromethanesulfonate

General Experimental Conditions

Intermediates and final compounds were purified by flash chromatographyusing one of the following instruments: i) Biotage SP1 system and theQuad 12/25 Cartridge module. ii) ISCO combi-flash chromatographyinstrument. Silica gel Brand and pore size: i) KP-SIL 60 Å, particlesize: 40-60 μm; ii) CAS registry NO: Silica Gel: 63231-67-4, particlesize: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang ChemicalCo., Ltd, pore: 200-300 or 300-400.

Intermediates and final compounds were purified by preparative HPLC onreversed phase column using X Bridge™ Perp C₁₈ (5 μm, OBD™ 30×100 mm)column or SunFire™ Perp C₁₈ (5 μm, OBD™ 30×100 mm) column.

LC/MS spectra were obtained using a Waters UPLC-SQD Mass. Standard LC/MSconditions were as follows (running time 3 minutes):

Acidic condition: A: 0.1% formic acid and 1% acetonitrile in H₂O; B:0.1% formic acid in acetonitrile;

-   -   Basic condition: A: 0.05% NH₃H₂O in H₂O; B: acetonitrile.    -   Mass spectra (MS): generally only ions which indicate the parent        mass are reported, and unless otherwise stated the mass ion        quoted is the positive mass ion (M+H)⁺.

NMR Spectra were obtained using Bruker Avance 400 MHz.

All reactions involving air-sensitive reagents were performed under anargon atmosphere. Reagents were used as received from commercialsuppliers without further purification unless otherwise noted.

PREPARATIVE EXAMPLES Example 15-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

Preparation ofO-[(3aR,5R,6S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]methylsulfanylmethanethioate

To a suspension of NaH (60% in mineral oil, 4 g, 100 mmol) in THF (80mL) was added a solution of diacetone-α-D-glucose (10.5 g, 40 mmol) andimidazole (136 mg, 2 mmol) in THF (20 mL) dropwise while keeping innertemperature below 15° C. The formed mixture was stirred at 10° C. for 15minutes. To the previous mixture was added carbon disulfide (14.8 g, 200mmol) and the reaction mixture was stirred at room temperature for 1hour. The reaction mixture was added iodomethane (24.6 g, 200 mmol) andstirred at room temperature for another 2 hours, then quenched bysaturated NH₄Cl solution (70 mL) and extracted with EtOAc (100 mL)twice. The combined organic layers were dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (eluting with 1:10 EtOAc in petroleum ether) to afford14.6 g ofO-[(3aR,5R,6S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]methylsulfanylmethanethioate (compound 1a) as a colorless oil.

Compound 1a: ¹H NMR (400 MHz, CDCl₃) δ ppm: 5.89-5.97 (m, 2H), 4.65-4.73(m, 1H), 4.29-4.39 (m, 2H), 4.04-4.17 (m, 2H), 2.61 (s, 3H), 1.56 (s,3H), 1.44 (s, 3H), 1.35 (d, J=4.02 Hz, 6H).

Preparation of(3aR,5S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole

To a solution ofO-[(3aR,5R,6S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]methyl sulfanylmethanethioate (compound 1a, 14 g, 40 mmol) in toluenewas added tri-n-butyltin hydride(23.2 g, 80 mmol) andazodiisobutyronitrile (82 mg, 0.5 mmol), the formed mixture was heatedat 130° C. under nitrogen for 3 hours. After the reaction was completed,the reaction mixture was concentrated in vacuo and the residue waspurified by column chromatography on silica gel (eluting with 1:10 EtOAcin petroleum ether) to afford 8.2 g of(3aR,5S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole(compound 1b) as an oil.

Compound 1b: ¹H NMR (400 MHz, CDCl₃) δ ppm: 5.82 (d, J=3.76 Hz, 1H),4.73-4.80 (m, 1H), 4.12 (m, 3H), 3.78-3.88 (m, 1H), 2.15-2.24 (m, 1H),1.73-1.83 (m, 1H), 1.52 (s, 3H), 1.43 (s, 3H), 1.36 (s, 3H), 1.32 (s,3H). MS obsd. (ESI⁺) [(M+NH₄)⁺]: 262.

Preparation of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethane-1,2-diol

A solution of(3aR,5S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole(compound 1b, 10.0 g, 40.9 mmol) in 60% HOAc in water (20 mL) wasstirred at 40° C. for 16 hours. The reaction mixture was adjusted to pH8-8.5 by saturated NaHCO₃ solution and extracted with EtOAc. The organiclayer was combined and concentrated, the residue was purified by columnchromatography on silica gel (eluting with 1:2 EtOAc in petroleum ether)to afford 5.2 g of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethane-1,2-diol(compound 1c). MS obsd. (ESI⁺) [(M+NH₄)⁺]: 222.

Preparation of(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde

To a solution of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]lethane-1,2-diol(compound 1c, 18 g, 90 mmol) in the MeOH (250 mL) cooled in ice bath wasadded sodium metaperiodate (23.1 g, 108 mmol). After being stirred atroom temperature for 12hours, the resulting suspension was filtered. Thefiltrate was concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 1:2 EtOAc in petroleum ether)to afford 14 g of(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde(compound 1d). MS obsd. (ESI⁺) [(M+NH₄)⁺]: 190.

Preparation of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol

To a solution of(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde(compound 1d, 296 mg, 2 mmol) in THF (20 mL) was added ethyl magnesiumbromide (2M in THF, 2 mL, 2 mmol) at −20° C. under argon. After beingstirred at −20° C. for 20 hours, the reaction was quenched by saturatedNH₄Cl solution and extracted with EtOAc (30 mL) three times. Thecombined organic layers were concentrated in vacuo to afford the crudeproduct of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 1e), which was used in next step without further purification.MS obsd. (ESI⁺) [(M+NH₄)⁺]: 316.

Preparation of[(3R,5S)-2-acetoxy-5-(1-acetoxypropyl)tetrahydrofuran-3-yl]acetate

To a solution of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 1e, crude as prepared above) in the mixture of acetic acid (2mL) and acetic acid anhydride (2 mL) was added H₂SO₄ (0.2 mmol). Afterbeing stirred at room temperature for 24 hours, the solution was dilutedby EtOAc (40 mL) and adjusted to pH 5.0 by saturated NaHCO₃ solution.The separated organic layer was washed with brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (eluting with 1:3 EtOAc in petroleum ether) to afford 510mg of [(3R,5S)-2-acetoxy-5-(1-acetoxypropyl)tetrahydrofuran-3-yl]acetate (compound 10. MS obsd. (ESI⁺) [(M+NH₄)⁺]: 316.

Preparation of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (CAS #:30161-97-8, Cat. #: as J92-094790, commercially available from J&KScientific, 276 mg, 1.5 mmol) in ACN (20 mL) was added BSA (913.5 mg,4.5 mmol). The reaction mixture was stirred at 70° C. for 0.5 hour underargon to form a clear solution. After the solution was cooled to roomtemperature, [(3R,5S)-2-acetoxy-5-(1-acetoxypropyl)tetrahydrofuran-3-yl]acetate (compound 1f, 450 mg, 1.6 mmol) and TMSOTf (510 mg, 2.3 mmol)were added in sequence. After being heated at 70° C. for 14 hours, thesolvent was evaporated in vacuo. The residue was partitioned betweenEtOAc and saturated NaHCO₃ solution (30 mL). The organic layer wascollected and the aqueous phase was extracted with EtOAc (30 mL) twice.The combined organic layers were washed with brine, dried over Na₂SO₄and concentrated in vacuo to afford 412 mg crude product of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate(compound 1g), which was used in next step without purification. MSobsd. (ESI⁻) [(M−H)⁻]: 411.

Preparation of5-amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 1g, crude, 412 mg) in methanol (25 mL) was added K₂CO₃(272 mg, 2 mmol). After being stirred at room temperature for 12 hours,the reaction mixture was adjusted to pH 8.2 by addition of HOAc (120 mg,2 mmol) and concentrated in vacuo. The residue was purified andseparated by preparative HPLC to afford 133.1 mg of Example 1-A and118.2 mg of Example 1-B as white solid.

Example 1-A: ¹NMR (400 MHz, CD₃OD) δ ppm: 5.93-6.00 (m, 1H), 4.91-4.94(m, 1H), 4.15-4.25 (m, 1H), 3.44-3.53 (m, 1H), 2.49-2.61 (m, 1H),1.89-1.96 (m, 1H), 1.41-1.61 (m, 2H), 1.01 (t, J=7.40 Hz, 3H). MS obsd.(ESI⁻) [(M−H⁻)⁻]: 327.

Example 1-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.89-5.96 (m, 1H), 4.90-4.97(m, 1H), 4.09-4.20 (m, 1H), 3.61-3.69 (m, 1H), 2.57-2.68 (m, 1H),1.91-1.96 (m, 1H), 1.55-1.65 (m, 1H), 1.35-1.46 (m, 1H), 1.00 (t, J=7.40Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 327.

Example 25-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one

Preparation of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate

To a suspension of 5-amino-3H-thiazolo[4,5-d]pyrimidin-2-one (CAS #:848691-22-5, Cat. #: as SY028954, commercially available from ShanghaiShaoyuan Co. Ltd., 326 mg, 2 mmol) in ACN (40 mL) was added BSA (1.2 g,6 mmol). The resulting reaction mixture was then stirred at 70° C. for 1hour under argon to form a clear solution. After the solution was cooledto room temperature,[(3R,5S)-2-acetoxy-5-(1-acetoxypropyl)tetrahydrofuran-3-yl] acetate(compound 1f, 432 mg, 1.5 mmol) and TMSOTf (666 mg, 3 mmol) were addedin sequence. After being heated with stirring at 70° C. for 14 hours,the solvent was evaporated in vacuo. The residue was partitioned betweenEtOAc and saturated NaHCO₃ solution (30 mL). The organic layer wasseparated and the aqueous phase was extracted with EtOAc (30 mL) twice.The combined organic layers were washed with brine, dried over Na₂SO₄and concentrated in vacuo to afford crude[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate. The crude product was purified by column chromatography onsilica gel (eluting with 1:1 EtOAc in petroleum ether) to afford 310 mgof[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 2a).

Preparation of5-amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one

To a solution of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 2a, 180 mg, 0.5 mmol) in methanol (25 mL) was addedK₂CO₃ (136 mg, 1 mmol). After being stirred at room temperature for 2hours, the reaction mixture was adjusted to pH 7.0 by addition of HOAc(120 mg, 2 mmol) and concentrated in vacuo. The residue was purified andseparated by preparative HPLC to afford 9.5 mg of Example 2-A and 2.8 mgof Example 2-B as white solid.

Example 2-A: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 8.35 (s, 1H), 6.85 (s,2H), 5.81-5.87 (m, 1H), 5.43-5.52 (m, 1H), 4.73-4.81 (m, 1H), 4.48-4.59(m, 1H), 3.95-4.05 (m, 1H), 3.27-3.32 (m, 1H), 2.31-2.41 (m, 1H),1.69-1.78 (m, 1H), 1.36-1.48 (m, 1H), 1.18-1.33 (m, 1H), 0.88 (t, J=7.40Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]: 313.

Example 2-B: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 8.35 (s, 1H), 6.84 (s,2H), 5.79-5.88 (m, 1H), 5.37-5.54 (m, 1H), 4.77-4.86 (m, 1H), 4.52-4.62(m, 1H), 3.87-4.01 (m, 1H), 3.30-3.34 (m, 1H), 2.39-2.49 (m, 1H), 1.86(ddd, J=2.76, 6.21, 12.61 Hz, 1H), 1.49 (ddd, J=3.26, 7.47, 13.61 Hz,1H), 1.14-1.28 (m, 1H), 0.86 (t, J=7.40 Hz, 3H). MS obsd. (ESI⁺)[(M+H)⁺]: 313.

Example 3[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(1-hydroxypropyl)tetrahydrofuran-3-yl]acetate

The title compound was prepared according to the following scheme:

Preparation of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propoxy-tert-butyl-diphenyl-silane

To a solution of 1-[(3aR,5S,6aR)-2,2-dimethyl-3 a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol (compound 1e,2.02 g, 10 mmol) in DMF (30 mL) was added imidazole (6.5 g, 100 mmol)and tert-butylchlorodiphenylsilane (8.22 g, 30 mmol) with stirring.After being stirred at room temperature for 2 hours, the resultingsolution was diluted by EtOAc (200 mL), washed with water, brine anddried over Na₂SO₄. The organic layer was concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (elutingwith 1:10 EtOAc in petroleum ether) to afford 3.6 g of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propoxy-tert-butyl-diphenyl-silane (compound 3a).

Compound 3a: ¹H NMR (400 MHz, CDCl₃) δ ppm: 7.65-7.79 (m, 4H), 7.33-7.49(m, 6H), 5.52-5.81 (m, 1H), 4.64-4.72 (m, 1H), 4.19-4.32 (m, 1H),3.67-4.01 (m, 1H), 1.98-2.05 (m, 1H), 1.74-1.94 (m, 1H), 1.61 (s, 6H),1.34-1.44 (m, 2H), 1.07 (d, J=1.25 Hz, 9H), 0.72-0.83 (m, 3H). MS obsd.(ESI⁺) [(M+NH₄)⁺]: 458.

Preparation of[(2R,3R,5S)-2-acetoxy-5-[1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-3-yl]acetate

To a solution of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propoxy-tert-butyl-diphenyl-silane(compound 3a, 3.6 g, 8.2 mmol) in the DCM (30 mL) was added acetic acid(15 mL), acetic acid anhydride (15 mL) and H₂SO₄ (0.8 mmol). After beingstirred at room temperature for 24 hours, TEA (5 mL) was added to thereaction mixture. The resulted solution was diluted with DCM (30 mL).Theseparated organic layer was washed with saturated NaHCO₃ solution, brineand dried over MgSO₄. The organic layer was concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (elutingwith 1:30 EtOAc in petroleum ether) to afford 3.7 g of[(2R,3R,5S)-2-acetoxy-5-[1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-3-yl]acetate (compound 3b). MS obsd. (ESI⁺) [(M+NH₄)⁺]: 502.

Preparation of[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-[1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-3-yl]acetate

To a suspension of 5-amino-3H-thiazolo[4,5-d]pyrimidin-2-one (1.08 g, 6mmol) in ACN (100 mL) was added BSA (3.6 g, 18 mmol). The reactionmixture was stirred at 70° C. for 1 hour under argon to form a clearsolution. After the solution was cooled to room temperature,[(2R,3R,5S)-2-acetoxy-5-[1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-3-yl]acetate (compound 3b, 1.45 g, 3 mmol) and TMSOTf (2.0 g, 9 mmol) wereadded in sequence. After being heated with stirring at 70° C. for 14hours, the solvent was removed in vacuo. The residue was partitionedbetween EtOAc (50 mL) and saturated NaHCO₃ solution (30 mL). The organiclayer was separated and the aqueous phase was extracted with EtOAc (50mL) twice. The combined organic layers were washed with brine, driedover Na₂SO₄ and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (eluting with 1:1 EtOAc in petroleumether) to afford 1.04 g of[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-[1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-3-yl]acetate (compound 3c). MS obsd. (ESI⁺) [(M+H⁺)⁺]: 593.

Preparation of[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(1-hydroxypropyl)tetrahydrofuran-3-yl]acetate

To a solution of[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-[1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-3-yl](compound 3c, 1.04 g, 1.8 mmol) in THF (20 mL) was added TBAF solution(1M in THF, 6 mL, 6 mmol) with stirring. After being stirred at roomtemperature for 4 hours, the reaction mixture was washed with saturatedNH₄Cl solution, dried over Na₂SO₄ and concentrated in vacuo. The residuewas purified by column chromatography on silica gel (eluting with 1:2EtOAc in petroleum ether) to afford 620 mg of[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-(1-hydroxypropyl)tetrahydrofuran-3-yl]acetate (Example 3), which was further purified and separated bypreparative HPLC to afford 112.8 mg of Example 3-A and 99.8 mg ofExample 3-B as white solid.

Example 3-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.22 (s, 1H), 6.04-6.07 (m,1H), 5.74-5.80 (m, 1H), 4.12-4.19 (m, 1H), 3.50-3.57 (m, 1H), 2.76 (ddd,J=7.40, 10.23, 13.49 Hz, 1H), 2.10 (s, 3H), 2.04-2.10 (m, 1H), 1.54-1.63(m, 1H), 1.42-1.52 (m, 1H), 1.02 (t, J=7.40 Hz, 3H). MS obsd. (ESI⁺)[(M+H)⁺]: 355.

Example 3-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.22 (s, 1H), 5.99-6.07 (m,1H), 5.70-5.81 (m, 1H), 4.06-4.18 (m, 1H), 3.61-3.71 (m, 1H), 2.77-2.90(m, 1H), 2.11-2.16 (m, 1H), 2.09 (s, 3H), 1.57-1.68 (m, 1H), 1.34-1.46(m, 1H), 1.01 (t, J=7.40 Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]: 355.

Example 41-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylacetate

To a solution of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 2a, 150 mg, 0.4 mmol) in methanol (25 mL) was addedK₂CO₃ (14 mg, 0.1 mmol). After being stirred at room temperature for 0.5hour, the reaction was adjusted to pH 7.0 by addition of HOAc (12.6 mg,0.2 mmol) and concentrated in vacuo. The residue was purified andseparated by preparative HPLC to afford 17.5 mg of Example 4-A and 8.5mg of Example 4-B as white solid.

Example 4-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.20 (s, 1H), 5.98-6.08 (m,1H), 4.93-5.01 (m, 2H), 4.31-4.42 (m, 1H), 2.56-2.70 (m, 1H), 2.03 (s,3H), 1.87-1.95 (m, 1H), 1.54-1.78 (m, 2H), 0.93 (t, J=7.53 Hz, 3H). MSobsd. (ESI⁻) [(M+H)⁺]: 355.

Example 4-B: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 8.28-8.39 (m, 1H),6.81-6.92 (br. s., 2H), 5.76-5.86 (m, 1H), 5.46-5.58 (br. s, 1H),4.92-5.02 (m, 1H), 4.79-4.89 (m, 1H), 4.14-4.23 (m, 1H), 2.42-2.48 (m,1H), 1.98 (s, 3H), 1.78-1.88 (m, 1H), 1.55-1.70 (m, 1H), 1.34-1.49 (m,1H), 0.82 (t, J=7.40 Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]: 355.

Example 5[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(1-hydroxypropyl)tetrahydrofuran-3-yl]acetate

To a solution of5-amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 1, 328 mg, 1 mmol) in DCM (15 mL) was added TEA (404 mg, 4mmol) and acetic anhydride (48 mg, 1 mmol) with stirring. After beingstirred at room temperature for 2 hours, the resulting solution wasquenched by acetic acid (240 mg, 4 mmol), washed with brine, dried overMgSO₄ and concentrated in vacuo. The residue was purified and separatedby preparative HPLC to afford 31.5 mg of Example 5-A and 20.0 mg ofExample 5-B as white solid.

Example 5-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.96 (d, J=2.51 Hz, 1H),5.63-5.77 (m, 1H), 4.05-4.17 (m, 1H), 3.47-3.55 (m, 1H), 2.62-2.79 (m,1H), 2.09 (s, 3H), 2.00-2.06 (m, 1H), 1.51-1.63 (m, 1H), 1.41-1.51 (m,1H), 1.02 (t, J=7.53 Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 369.

Example 5-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.95 (d, J=2.51 Hz, 1H),5.68-5.77 (m, 1H), 4.04-4.14 (m, 1H), 3.60-3.69 (m, 1H), 2.73-2.84 (m,1H), 2.09 (s, 4H), 1.57-1.67 (m, 1H), 1.35-1.45 (m, 1H), 1.01 (t, J=7.40Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 369.

Example 6[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]acetate

The title compound was prepared according to the following scheme:

Preparation of[(2R)-2-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-hydroxy-ethyl]4-methylbenzenesulfonate

To a solution of(1R)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethane-1,2-diol(100 g, 490 mmol) in dry pyridine (1000 mL) was added p-toluenesulfonylchloride (139 g, 735 mmol) at 0° C. After being stirred at roomtemperature for 12 hours, the resulted solution was quenched by water(100 mL) and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 1:10 to 1:3 EtOAc inpetroleum ether) to afford 130 g of[(2R)-2-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-hydroxy-ethyl]4-methylbenzenesulfonate (compound 6a) as a slight yellow oil.

Compound 6a: ¹H NMR (400 MHz, CDCl₃) δ ppm: 7.82 (d, J=8.00 Hz, 2H),7.38 (d, J=8.00 Hz, 2H), 5.78 (d, J=3.76 Hz, 1H), 4.75 (t, J=4.00 Hz,1H), 4.20-4.12 (m, 2H), 4.03-3.97 (m, 2H), 2.48 (s, 3H), 2.39 (d, J=3.51Hz, 1H), 2.08-2.15 (m, 1H), 1.75-1.80 (m, 1H), 1.51 (s, 3H), 1.33 (s,3H).

Preparation of(3aR,5S,6aR)-2,2-dimethyl-5-[(2R)-oxiran-2-yl]-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole

To a solution of[(2R)-2-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-hydroxy-ethyl]4-methylbenzenesulfonate (compound 6a, 100 g, 280 mmol) in anhydrous THF(1500 mL) cooled at 31 70° C. was added potassiumbis(trimethylsilyl)amide (340 mL, 340 mmol, 1 M in THF) under N₂atmosphere. After being stirred at −70° C. for 1 hour, the reactionmixture was poured into saturated NH₄Cl solution. The organic layer wasseparated and the aqueous phase was extracted with EtOAc. The combinedorganic layers were dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (elutingwith 1:3 EtOAc in petroleum ether) to afford 40.5 g of(3aR,5S,6aR)-2,2-dimethyl-5-[(2R)-oxiran-2-yl]-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole(compound 6b) as a slight yellow oil.

Compound 6b: ¹H NMR: (400 MHz, CDCl₃) δ ppm: 5.87 (d, J=3.76 Hz, 1H),4.77 (t, J=4.00, 1H), 4.20-4.28 (m, 1H), 3.14-3.20 (m, 1H), 2.83-2.88(m, 1H), 2.63 (dd, J=5.00, 2.80 Hz, 1H), 2.09 (dd, J=12.00, 4.00 Hz,1H), 1.69-1.79 (m, 1H), 1.52 (s, 3H), 1.34 (s, 3H).

Preparation of(1R)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol

To a suspension of CuI (19.3 g, 107 mmol) in dry THF (2000 mL) under N₂atmosphere was added methyl magnesium bromide (3 M in diethyl ether, 537mL, 1.61 mol) at −70° C. After being stirred at this temperature for 1hour, a solution of(3aR,5S,6aR)-2,2-dimethyl-5-[(2R)-oxiran-2-yl]-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole(compound 6b, 100 g, 537 mmol, dissolved in anhydrous THF 200 mL) wasadded to reaction mixture dropwise. After being stirred at −70° C. foradditional 2 hours, the reaction mixture was poured into saturated NH₄Clsolution. The organic layer was separated and the aqueous phase wasextracted with EtOAc twice. The combined organic layers were dried overNa₂SO₄ and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 1:3 EtOAc in petroleum ether)to afford 82 g of(1R)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 6c) as a slight yellow solid.

Compound 6c: ¹H NMR (400 MHz, CDCl₃) δ ppm: 5.83 (d, J=3.76 Hz, 1H),4.81-4.73 (m, 1H), 4.26-4.19 (m, 1H), 3.91-3.82 (m, 1H), 2.08-2.02 (m,1H), 1.93-1.89 (m, 1H), 1.54 (s, 3H), 1.49-1.39 (m, 2H), 1.34 (s, 3H),1.02 (t, J=7.53 Hz, 3H).

Preparation of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]4-nitrobenzoate

To a stirred solution of(1R)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 6c, 50 g, 245 mmol), triphenylphosphine (195 g, 743 mmol),4-nitrobenzoic acid (124 g, 743 mmol) in THF (1200 mL) was added diethylazodicarboxylate (130 g, 743 mmol) dropwise at 0° C. under N₂. Afterbeing stirred at 18° C. for 10 hours, the mixture was quenched byaddition of saturated NaHCO₃ solution and extracted with EtOAc. Theorganic layers were combined, dried over Na₂SO₄ and concentrated invacuo. The residue was purified by column chromatography on silica gel(eluting with 1:3 EtOAc in petroleum ether) to afford 61 g of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]4-nitrobenzoate (compound 6d) as a slight yellow solid.

Compound 6d: ¹H NMR (400 MHz, CDCl₃) δ ppm: 8.34-8.22 (m, 4H), 5.85 (d,J=3.76 Hz, 1H), 5.23-5.17 (m, 1H), 4.76 (t, J=4.27 Hz, 1H), 4.48-4.39(m, 1H), 2.12 (dd, J=13.30, 4.52 Hz, 1H), 1.88-1.78 (m, 2H), 1.71-1.62(m, 1H), 1.55 (s, 3H), 1.34 (s, 3H), 1.01 (t, J=7.40 Hz, 3H).

Preparation of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol

To a solution of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]4-nitrobenzoate (compound 6d, 100 g, 285 mmol) in methanol (1200 mL) wasadded K₂CO₃ (78.7 g, 570 mmol). After being stirred at room temperaturefor 10 minutes, the resulted mixture was filtered. The filtrate wasconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (eluting with 1:8 EtOAc in petroleum ether) to afford 54.7g of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 6e) as a slight yellow solid.

Compound 6e: ^(1-H NMR ()400 MHz, CDCl₃) δ ppm: 5.81 (d, J=3.64 Hz, 1H),4.75 (t, J=4.20 Hz, 1H), 4.18-4.11 (m, 1H), 3.49-3.40 (m, 1H), 2.07-2.00(m, 2H), 1.84-1.75 (m, 1H), 1.59-1.47 (m, 5H), 1.32 (s, 3H), 1.01 (t,J=7.40 Hz, 3H).

Preparation of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]acetate

To a stirred solution of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 6e,13.5 g, 67 mmol), TEA (81 g, 804 mmol), DMAP (1.6 g, 13mmol) in anhydrous DCM (150 mL) was added acetic anhydride (62 g, 603mmol). After being stirred at 22° C. for 10 hours, the reaction wasquenched by the saturated NaHCO₃ solution. The organic layer wasseparated and the aqueous phase was extracted with EtOAc. The combinedorganic layers were dried over Na₂SO₄, and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (elutingwith 1:8 EtOAc in petroleum ether) to afford 13 g of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]acetate (compound 6f) as a colourless oil.

Compound 6f: ¹H NMR (400 MHz, CDCl₃) δ ppm: 5.83 (d, J=3.76 Hz, 1H),4.92 (dt, J=7.97, 5.18 Hz, 1H), 4.74 (t, J=4.00 Hz, 1H), 4.35-4.27 (m,1H), 2.12 (s, 3H), 2.08-1.99 (m, 1H), 1.74-1.56 (m, 3H), 1.53 (s, 3H),1.34 (s, 3H), 0.95 (t, J=7.40 Hz, 3H).

Preparation of[(3R,5S)-2-acetoxy-5-[(1S)-1-acetoxypropyl]tetrahydrofuran-3-yl]acetate

To a solution of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]acetate (compound 6f, 4.8 g, 20 mmol), acetic acid (12.2 g, 200 mmol)and acetic anhydride (10.2 g, 100 mmol) in anhydrous DCM (100 mL) wasadded concentrated H₂SO₄ (0.5 mL) at 0° C. After being stirred at 22° C.for 3 hours, the reaction was quenched by addition of saturated NaHCO₃solution. The organic layer was separated and the aqueous phase wasextracted with EtOAc. The combined organic layers were dried overNa₂SO₄, filtered, and concentrated in vacuo. The residue was purified bycolumn on silica gel (eluting with 1:8 EtOAc in petroleum ether) toafford 2.3 g of[(3R,5S)-2-acetoxy-5-[(1S)-1-acetoxypropyl]tetrahydrofuran-3-yl] acetate(compound 6g) as a colourless oil.

Compound 6g: ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.12 (s, 1H), 5.19 (d,J=4.52 Hz, 1H), 4.83-4.91 (m, 1H), 4.34-4.44 (m, 1H), 2.09-2.19 (m, 9H),1.51-1.74 (m, 4H), 0.94 (t, J=7.40 Hz, 3H).

Preparation of[(2R,3R,5S)-5-[(1S)-1-acetoxypropyl]-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (1.4 g, 7.5 mmol)in ACN (20 mL) was added BSA (7.7 g, 38 mmol). The reaction mixture wasstirred at 70° C. for 0.5 hour under argon to form a clear solution.After the solution was cooled to room temperature,[(3R,5S)-2-acetoxy-5-[(1S)-1-acetoxypropyl]tetrahydrofuran-3-yl] acetate(compound 6g,720 mg, 2.5 mmol) and TMSOTf (8.3 g, 38 mmol) were added insequence. After being heated with stirring at 70° C. for 14 hours, thesolvent was removed in vacuo. The residue was partitioned between EtOAcand saturated NaHCO₃ solution (30 mL). The organic layer was separatedand the aqueous phase was extracted with EtOAc (30 mL) twice. Thecombined organic layers were washed with brine, dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel to afford 470 mg of[(2R,3R,5S)-5-[(1S)-1-acetoxypropyl]-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 6h) as slight yellow solid. MS obsd. (ESI⁻) [(M−H)⁻]:411.

Preparation of[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]acetate

To a solution of[(2R,3R,5S)-5-[(1S)-1-acetoxypropyl]-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 6h, 210 mg, 0.5 mmol) in methanol (25 mL) was addedK₂CO₃ (136 mg, 1 mmol). After being stirred at room temperature for 10min, the reaction was adjusted to pH 7.0 by addition of HOAc (120 mg, 2mmol), concentrated in vacuo and the residue was purified by preparativeHPLC to afford 66.7 mg of[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]acetate (Example 6) as a white solid.

Example 6: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.94 (d, J=1.51 Hz, 1H),5.00-4.93 (m, 2H), 4.37-4.30 (m, 1H), 2.63-2.54 (m, 1H), 2.05 (s, 3H),1.91-1.83 (m, 1H), 1.74-1.58 (m, 2H), 0.93 (t, J=7.40 Hz, 3H). MS obsd.(ESI⁻) [(M−H)⁻]: 369.

Example 75-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by using methylmagnesium bromide instead of ethyl magnesium bromide. Example 7 waspurified and separated by preparative HPLC to afford Example 7-A andExample 7-B as white solid.

Example 7-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.91-5.97 (m, 1H), 4.91-4.96(m, 1H), 4.07-4.13 (m, 1H), 3.86-3.95 (m, 1H), 2.58-2.68 (m, 1H),1.92-2.01 (m, 1H), 1.17 (d, J=6.53 Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]:313.

Example 7-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.97 (d, J=2.76 Hz, 1H),4.90-4.94 (m, 1H), 4.06-4.14 (m, 1H), 3.73-3.82 (m, 1H), 2.46-2.58 (m,1H), 1.86-1.96 (m, 1H), 1.17 (d, J=6.27 Hz, 3H). MS obsd. (ESI⁻)[(M−H)⁻]: 313.

Example 85-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybut-3-enyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by using allylmagnesium bromide instead of ethyl magnesium bromide. Example 8 waspurified and separated by preparative HPLC to afford Example 8-A andExample 8-B as white solid.

Example 8-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.94-5.99 (m, 1H), 5.86-5.92(m, 1H), 5.05-5.15 (m, 3H), 4.18-4.26 (m, 1H), 3.64 (m, 1H), 2.51-2.60(m, 1H), 2.19-2.34 (m, 2H), 1.95 (m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 339.

Example 8-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.83-5.99 (m, 2H), 4.96-5.21(m, 3H), 4.17 (d, J=5.02 Hz, 1H), 3.80 (d, J=3.76 Hz, 1H), 2.58-2.73 (m,1H), 2.27-2.38 (m, 1H), 2.19 (td, J=7.06, 14.24 Hz, 1H), 1.89-2.01 (m,1H). MS obsd. (ESI⁻) [(M−H)⁻]: 339.

Example 95-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxypentyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by using butylmagnesium bromide instead of ethyl magnesium bromide. Example 9 waspurified and separated by preparative HPLC to afford Example 9-A andExample 9-B as white solid.

Example 9-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.95 (d, J=3.26 Hz, 1H),4.95-5.01 (m, 1H), 4.16-4.22 (m, 1H), 3.51-3.60 (m, 1H), 2.49-2.58 (m,1H), 1.90-2.00 (m, 1H), 1.44-1.55 (m, 3H), 1.20-1.40 (m, 3H), 0.87-0.98(m, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 355.

Example 9-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.90-5.95 (m, 1H), 4.93-4.99(m, 1H), 4.12-4.20 (m, 1H), 3.69-3.77 (m, 1H), 2.59-2.67 (m, 1H),1.90-1.98 (m, 1H), 1.49-1.60 (m, 2H), 1.29-1.44 (m, 4H), 0.91-0.97 (m,3H). MS obsd. (ESI⁻) [(M−H)⁻]: 355.

Example 105-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybutyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by using propylmagnesium bromide instead of ethyl magnesium bromide. Example 10 waspurified and separated by preparative HPLC to afford Example 10-A andExample 10-B as white solid.

Example 10-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.93-5.99 (m, 1H),4.92-4.95 (m, 1H), 4.13-4.22 (m, 1H), 3.53-3.63 (m, 1H), 2.48-2.60 (m,1H), 1.87-1.99 (m, 1H), 1.50-1.61 (m, 1H), 1.36-1.50 (m, 3H), 0.91-1.01(m, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 341.

Example 10-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.90-5.96 (m, 1H),4.92-4.96 (m, 1H), 4.11-4.19 (m, 1H), 3.71-3.80 (m, 1H), 2.56-2.69 (m,1H), 1.89-1.99 (m, 1H), 1.46-1.60 (m, 2H), 1.34-1.45 (m, 2H), 0.96 (t,J=6.90 Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 341.

Example 115-Amino-3-[(2R,3R,5S)-5-[cyclopentyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

Preparation of[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-cyclopentyl-methanol

To a solution of(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde(4.0 g, 23.2 mmol) in THF (20 mL) was added cyclopentylmagnesium bromide(1M in THF, 30 mL, 30 mmol) at −20° C. under argon. After being stirredat −20° C. for 20 hours, the reaction was quenched by saturated NH₄Clsolution. The reaction mixture was extracted with EtOAc (30 mL) threetimes. The organic layers were combined and concentrated in vacuo toafford 1.2 g crude product of[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-cyclopentyl-methanol(compound 11a) as a colorless oil, which was used in next step withoutfurther purification. MS obsd. (ESI⁺) [(M+H)⁺]: 243.

Preparation of[[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-cyclopentyl-methyl]benzoate

To a solution of[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-cyclopentyl-methanol(compound 11a, 1.2 g, 5.0 mmol), TEA (3.2 g, 31.2 mmol) and DMAP (100mg) in DCM (50 mL) was added benzoyl chloride (1.4 g, 10.0 mmol) slowlyat 0° C. The mixture was stirred at 25° C. for 4 hours and then quenchedby saturated NaHCO₃ solution. The reaction mixture was extracted withEtOAc (100 mL) twice. The organic layers were combined, washed withbrine (50 mL), dried over Na₂SO₄ and concentrated in vacuo. The residuewas purified by column chromatography on silica gel (eluting with 1:20to 1:5 EtOAc in petroleum ether) to afford 1.4 g of[[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-cyclopentyl-methyl]benzoate (compound 11b) as a colourless oil.

Preparation of[cyclopentyl-[(2S,4R)-4,5-diacetoxytetrahydrofuran-2-yl]methyl]benzoate

To a solution of[[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-cyclopentyl-methyl]benzoate (compound 11b, 800 mg, 2.3 mmol) in the mixture of acetic acid(2 mL) and acetic acid anhydride (2 mL) was added H₂SO₄ (0.2 mmol).After being stirred at room temperature for 24 hours, the solution wasdiluted by EtOAc (40 mL) and adjusted to pH 5.0 by addition of saturatedNaHCO₃ solution. The organic layer was separated, washed with brine,dried over Na₂SO₄ and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (eluting with 1:3 EtOAc in petroleumether) to afford 480 mg of[cyclopentyl-[(2S,4R)-4,5-diacetoxytetrahydrofuran-2-yl]methyl] benzoate(compound 11c).

Preparation of[[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]-cyclopentyl-methyl]benzoate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (370 mg, 2.0mmol) in ACN (20 mL) was added BSA (2.1 g, 10 mmol). The resultingreaction mixture was then stirred at 70° C. for 0.5 hour under argon toform a clear solution. After the solution was cooled to roomtemperature,[cyclopentyl-[(2S,4R)-4,5-diacetoxytetrahydrofuran-2-yl]methyl] benzoate(compound 11c, 400 mg, 1.0 mmol) and TMSOTf (2.25 g, 10 mmol) were addedin sequence. After being heated with stirring at 70° C. for 14 hours,the solvent was evaporated in vacuo. The residue was partitioned betweenEtOAc and saturated NaHCO₃ solution (30 mL). The organic layer wascollected and the aqueous phase was extracted with EtOAc (30 mL) twice.The combined organic layers were washed with brine, dried over Na₂SO₄and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 1:1 EtOAc in petroleum ether)to afford 160 mg of[[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]-cyclopentyl-methyl]benzoate (compound 11d).

Compound 11d: ¹H NMR (400 MHz, CDCl₃) δ ppm: 7.96-7.99 (m, 2H),7.59-7.61 (m, 1H), 7.44-7.50 (m, 2H), 5.82-5.93 (m, 1H), 5.23-5.26 (m,1H),4.45-4.52 (m, 1H), 3.73-3.76 (m, 1H), 2.81-2.85 (m, 1H), 2.41-2.43(m, 1H), 2.09 (s, 3H), 1.31-1.89 (m, 8H).

Preparation of5-amino-3-[(2R,3R,5S)-5-[cyclopentyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of[[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]-cyclopentyl-methyl]benzoate (compound 11d, 70 mg, 0.14 mmol) in methanol (10 mL) was addedK₂CO₃ (136 mg, 1 mmol). After being stirred at room temperature for 12hours, the reaction mixture was adjusted to pH 7.0 by addition of HOAc(120 mg, 2 mmol), concentrated in vacuo and the residue was purified bypreparative HPLC to afford 4.7 mg of5-amino-3-[(2R,3R,5S)-5-[cyclopentyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 11) as a white solid.

Example 11: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.91-5.93 (m, 1H), 4.94-4.98(m, 2H), 4.31-4.36 (m, 1H), 2.56-2.61 (m, 1H), 2.00-2.06 (m, 2H),1.31-1.72 (m, 8H). MS obsd. (ESI⁺) [(M+H)⁺]: 369.

Example 125-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxy-2-phenyl-ethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by using benzylmagnesium bromide instead of ethyl magnesium bromide. Example 12 waspurified and separated by preparative HPLC to afford Example 12-A andExample 12-B as white solid.

Example 12-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 7.24-7.33 (m, 4H),7.16-7.24 (m, 1H), 5.93-5.98 (m, 1H), 4.94-4.97 (m, 1H), 4.22 (dt,J=4.02, 7.53 Hz, 1H), 3.76-3.84 (m, 1H), 2.74-2.90 (m, 2H), 2.60 (td,J=7.53, 13.05 Hz, 1H), 1.97 (m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 389.

Example 12-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 7.51-7.57 (m, 1H),7.09-7.25 (m, 4H), 5.91-5.96 (m, 1H), 5.10-5.15 (m, 1H), 4.93-5.00 (m,2H), 4.39-4.48 (m, 1H), 2.74-2.87 (m, 1H), 2.28-2.35 (m, 2H), 1.82-1.92(m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 389.

Example 135-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxy-3-methyl-butyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by usingiso-butyl magnesium bromide instead of ethyl magnesium bromide. Example13 was purified and separated by preparative HPLC to afford Example 13-Aand Example 13-B as white solid.

Example 13-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.93-5.99 (m, 1H),4.91-4.95 (m, 1H), 4.10-4.19 (m, 1H), 3.62-3.69 (m, 1H), 2.48-2.59 (m,1H), 1.81-1.98 (m, 2H), 1.41-1.52 (m, 1H), 1.15-1.25 (m, 1H), 0.95 (t,J=6.78 Hz, 6H). MS obsd. (ESI⁻) [(M−H)⁻]: 355.

Example 13-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.89-5.96 (m, 1H),4.92-4.98 (m, 1H), 4.08-4.17 (m, 1H), 3.81-3.89 (m, 1H), 2.58-2.69 (m,1H), 1.89-1.99 (m, 1H), 1.78-1.89 (m, 1H), 1.23-1.40 (m, 2H), 0.94 (dd,J=6.65, 14.18 Hz, 6H). MS obsd. (ESI⁻) [(M−H)⁻]: 355.

Example 145-Amino-3-[(2R,3R,5S)-5-[cyclopropyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

Preparation of[(3R,5S)-2-acetoxy-5-[acetoxy(cyclopropyl)methyl]tetrahydrofuran-3-yl]acetate

Compound 14a was prepared in analogy to[(3R,5S)-2-acetoxy-5-(1-acetoxypropyl)tetrahydrofuran-3-yl] acetate(compound 1f), by using cyclopropyl magnesium bromide instead of ethylmagnesium bromide.

Preparation of[(2R,3R,5S)-5-[acetoxy(cyclopropyl)methyl]-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate

Compound 14b was prepared in analogy to[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 1g), by using[(3R,5S)-2-acetoxy-5-[acetoxy(cyclopropyl)methyl]tetrahydrofuran-3-yl]acetate (compound 14a) instead of[(3R,5S)-2-acetoxy-5-(1-acetoxypropyl)tetrahydrofuran-3-yl] acetate(compound 1f). MS obsd. (ESI⁻) [(M−H)⁻]: 441.

Preparation of5-amino-3-[(2R,3R,5S)-5-[cyclopropyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by using[(2R,3R,5S)-5-[acetoxy(cyclopropyl)methyl]-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 14b) instead of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 1g). Example 14 was purified and separated bypreparative HPLC to afford Example 14-A and Example 14-B as white solid.

Example 14-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.94-6.00 (m, 1H),4.93-4.96 (m, 1H), 4.27-4.35 (m, 1H), 2.91-2.98 (m, 1H), 2.54-2.66 (m,1H), 1.98-2.06 (m, 1H), 0.88-0.99 (m, 1H), 0.46-0.56 (m, 2H), 0.26-0.39(m, 2H). MS obsd. (ESI⁻) [(M−H)⁻]: 339.

Example 14-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.93-5.96 (m, 1H),4.92-5.00 (m, 1H), 4.30-4.38 (m, 1H), 3.09-3.16 (m, 1H), 2.68-2.79 (m,1H), 1.94-2.05 (m, 1H), 0.81-0.92 (m, 1H), 0.49-0.58 (m, 2H), 0.35-0.43(m, 1H), 0.25-0.33 (m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 339.

Example 15[[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]-cyclopropyl-methyl]acetate

To a solution of[(2R,3R,5S)-5-[acetoxy(cyclopropyl)methyl]-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 14b, crude, 220 mg, 0.5 mmol) in methanol (25 mL) wasadded K₂CO₃ (136 mg, 1 mmol). After being stirred at room temperaturefor 1 hour, the reaction was adjusted to pH 7.0 by addition of HOAc (120mg, 2 mmol), concentrated in vacuo and the residue was purified andseparated by preparative HPLC to afford 7.5 mg of Example 15-A and 7.5mg of Example 15-B as white solid.

Example 15-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.94-5.98 (m, 1H),4.83-4.87 (m, 1H), 4.39-4.47 (m, 2H), 2.62-2.70 (m, 1H), 1.92-2.07 (m,4H), 1.03-1.12 (m, 1H), 0.60-0.66 (m, 1H), 0.38-0.55 (m, 3H). MS obsd.(ESI⁻) [(M−H)⁻]: 381.

Example 15-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.89-5.96 (m, 1H),4.94-4.99 (m, 1H), 4.60-4.67 (m, 1H), 4.37-4.45 (m, 1H), 2.75-2.88 (m,1H), 2.04 (s, 3H), 1.90-2.00 (m, 1H), 0.98-1.08 (m, 1H), 0.58-0.66 (m,1H), 0.46-0.53 (m, 1H), 0.36 (m, 2H). MS obsd. (ESI⁻) [(M−H)⁻]: 381.

Example 16[(S)-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]-cyclopropyl-methyl]acetate

Preparation of[(2R,3R,5S)-5-[acetoxy(cyclopropyl)methyl]-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate(16a)

Compound 16a was prepared in analogy to[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 2a), by using[(3R,5S)-2-acetoxy-5-[acetoxy(cyclopropyl)methyl]tetrahydrofuran-3-yl]acetate (compound 14a) instead of[(3R,5S)-2-acetoxy-5-(1-acetoxypropyl)tetrahydrofuran-3-yl] acetate(compound 1f).

Preparation of[(S)-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]-cyclopropyl-methyl]acetate

The title compound was prepared in analogy to Example 4, by using[(2R,3R,5S)-5-[acetoxy(cyclopropyl)methyl]-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 16a) instead of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 2a). Example 16 was purified and separated bypreparative HPLC to afford Example 16-A and Example 16-B as white solid.

Example 16-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.21 (s, 1H), 6.06 (d,J=1.51 Hz, 1H), 4.93-4.98 (m, 1H), 4.38-4.52 (m, 2H), 2.64-2.76 (m, 1H),2.02 (s, 3H), 1.96-2.00 (m, 1H), 1.02-1.12 (m, 1H), 0.59-0.67 (m, 1H),0.49-0.55 (m, 1H), 0.36-0.48 (m, 2H). MS obsd. (ESI⁻) [(M+H)⁺]: 367.

Example 16-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.18-8.25 (m, 1H),5.98-6.07 (m, 1H), 4.96-5.03 (m, 1H), 4.64-4.72 (m, 1H), 4.40-4.51 (m,1H), 2.80-2.91 (m, 1H), 2.03 (s, 3H), 1.94-2.00 (m, 1H), 0.96-1.09 (m,1H), 0.58-0.68 (m, 1H), 0.44-0.55 (m, 1H), 0.27-0.41 (m, 2H). MS obsd.[(M+H)⁺]: 367.

Example 175-Amino-3-[(2R,3R,5S)-5-[cyclopropyl(hydroxy)methyl]-3-hydroxy-tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one

The title compound was prepared in analogy to Example 2, by using[(2R,3R,5S)-5-[acetoxy(cyclopropyl)methyl]-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 16a) instead of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 2a). Example 17 was purified and separated bypreparative HPLC to afford Example 17-A and Example 17-B as white solid.

Example 17-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.22 (s, 1H), 6.07 (d,J=3.01 Hz, 1H), 4.93-4.98 (m, 1H), 4.29-4.40 (m, 1H), 2.93-3.01 (m, 1H),2.59-2.69 (m, 1H), 2.00-2.09 (m, 1H), 0.89-0.98 (m, 1H), 0.49-0.58 (m,2H), 0.32-0.41 (m, 2H). MS obsd. (ESI⁻) [(M+H)⁺]: 325.

Example 17-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.23 (s, 1H), 5.99-6.06 (m,1H), 4.96-5.02 (m, 1H), 4.33-4.40 (m, 1H), 3.10-3.17 (m, 1H), 2.74-2.81(m, 1H), 2.00-2.07 (m, 1H), 0.83-0.92 (m, 1H), 0.49-0.58 (m, 2H),0.36-0.42 (m, 1H), 0.26-0.33 (m, 1H). MS obsd. [(M+H)⁺]: 325.

Example 185-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyprop-2-ynyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by usingethynyl magnesium bromide instead of ethyl magnesium bromide. Example 18was purified and separated by preparative HPLC to afford Example 18-Aand Example 18-B as white solid.

Example 18-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 6.00 (d, J=2.51 Hz, 1H),4.90-4.98 (m, 1H), 4.35-4.42 (m, 1H), 4.22-4.33 (m, 1H), 2.56-2.63 (m,1H), 1.97-2.11 (m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 323.

Example 18-B: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.26-11.41 (br. s, 1H),6.90-7.07 (br. s, 2H), 5.71-5.77 (m, 1H), 5.56-5.64 (m, 1H), 5.44-5.50(m, 1H), 4.78-4.86 (m, 1H), 4.16-4.23 (m, 1H), 4.02-4.13 (m, 1H),2.41-2.47 (m, 1H), 1.80-1.92 (m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 323.

Example 195-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxybut-2-ynyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by usingpropynylmagnesium bromide instead of ethyl magnesium bromide. Example 19was purified and separated by preparative HPLC to afford Example 19-Aand Example 19-B as white solid.

Example 19-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.97-6.03 (m, 1H),4.92-4.97 (m, 1H), 4.32-4.37 (m, 1H), 4.22-4.29 (m, 1H), 2.57-2.66 (m,1H), 1.99-2.07 (m, 1H), 1.84 (d, J=2.26 Hz, 3H). MS obsd. (ESI⁻)[(M−H)⁻]: 337.

Example 19-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.93-5.98 (m, 1H),4.95-5.01 (m, 1H), 4.41-4.46 (m, 1H), 4.24-4.32 (m, 1H), 2.67-2.77 (m,1H), 1.98-2.07 (m, 1H), 1.83 (d, J=2.01 Hz, 3H). MS obsd. (ESI⁻)[(M−H)⁻]: 337.

Example 205-Amino-3-[(2R,3R,5S)-3-hydroxy-5-[hydroxy(2-thienyl)methyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by using2-thienyl lithium instead of ethyl magnesium bromide. Example 20 waspurified and separated by preparative HPLC to afford Example 20-A andExample 20-B as white solid.

Example 20-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 7.33-7.39 (m, 1H),7.07-7.11 (m, 1H), 6.98-7.02 (m, 1H), 6.02-6.06 (m, 1H), 4.90-4.97 (m,2H), 4.46-4.52 (m, 1H), 2.52-2.57 (m, 1H), 1.71-1.76 (m, 1H). MS obsd.(ESI⁻) [(M−H)⁻]: 381.

Example 20-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 7.30-7.34 (m, 1H),7.02-7.05 (m, 1H), 6.98 (d, J=5.02 Hz, 1H), 5.96 (d, J=3.76 Hz, 1H),5.09-5.14 (m, 1H), 4.98-5.04 (m, 1H), 4.43-4.49 (m, 1H), 2.69-2.77 (m,1H), 1.94-2.02 (m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 381.

Example 215-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxy-2-methoxy-ethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared according to the following scheme:

Preparation of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanol

To a stirred solution of[(2S)-2-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-hydroxy-ethyl]4-methylbenzenesulfonate (compound 6a, 3.2 g, 8.9 mmol) in methanol (50mL) was added K₂CO₃ (5.4 g, 40 mmol). After being stirred at roomtemperature for 2 hours, the resulting solution was concentrated invacuo and the residue was purified by column chromatography on silicagel (eluting with 1:10 EtOAc in petroleum ether) to afford 1.62 g of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanol(compound 21b) as a colorless oil.

Compound 21b: ¹H NMR (400 MHz, CDCl₃) δ ppm: 5.76-5.83 (m, 1H),4.67-4.77 (m, 1H), 4.15-4.25 (m, 1H), 3.90-4.00 (m, 1H), 3.46 (d, J=3.76Hz, 1H), 3.31-3.42 (m, 4H), 2.57-2.68 (m, 1H), 2.01-2.10 (m, 1H),1.78-1.90 (m, 1H), 1.49 (s, 3H), 1.31 (s, 3H).

Preparation of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanone

To a solution of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanol(compound 21b, 1.62 g, 7.4 mmol) in DCM (50 mL) was added Dess-Martineperiodinane (4.7 g, 11 mmol) with stirring. After being stirred at roomtemperature for 2 hours, the resulting solution was filtered and thefiltrate was concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 1:2 EtOAc in petroleum ether)to afford 1.4 g of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanone(compound 21c) as a colorless oil. MS obsd. (ESI⁺) [(M+NH₄)⁺]: 234.

Preparation of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanol

To a stirred solution of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanone(compound 21c, 1.4 g, 6.5 mmol) in methanol (50 mL) was added sodiumborohydride (494 mg, 13 mmol). After being stirred at room temperaturefor 2 hours, the resulting solution was quenched by saturated NH₄Clsolution and concentrated in vacuo. The residue was suspended in EtOAcand then filtered. The filtrate was concentrated in vacuo to afford 1.24g of crude product of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanol(compound 21d) as a colorless oil. MS obsd. (ESI⁺) [(M+NH₄)⁺]: 236.

Preparation of[(2R,3R,5S)-2-acetoxy-5-(1-acetoxy-2-methoxy-ethyl)tetrahydrofuran-3-yl]acetate

To a solution of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-2-methoxy-ethanol(compound 21d, 1.24 g, 5.7 mmol) in the mixture of acetic acid (4 mL)and acetic acid anhydride (4 mL) was added H₂SO₄ (0.3 mmol). After beingstirred at room temperature for 24 hours, the solution was diluted byEtOAc (40 mL) and adjusted to pH 8.0 by addition of saturated NaHCO₃solution. The organic layer was separated, washed with brine, dried overNa₂SO₄, concentrated in vacuo and the residue was purified by columnchromatography on silica gel (eluting with 1:2 EtOAc in petroleum ether)to afford 1.5 g of[(2R,3R,5S)-2-acetoxy-5-(1-acetoxy-2-methoxy-ethyl)tetrahydrofuran-3-yl]acetate (compound 21e) as a colorless oil. MS obsd. (ESI⁺) [(M+NH₄)⁺]:322.

Preparation of[(2R,3R,5S)-5-(1-acetoxy-2-methoxy-ethyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (368 mg, 2.0mmol) in ACN (20 mL) was added BSA (1.2 mg, 6.0 mmol). The reactionmixture was stirred at 70° C. for 0.5 hour under argon to form a clearsolution. After the solution was cooled to room temperature,[(2R,3R,5S)-2-acetoxy-5-(1-acetoxy-2-methoxy-ethyl)tetrahydrofuran-3-yl]acetate (compound 21e, 304 mg, 1.0 mmol) and TMSOTf (666 mg, 3.0 mmol)were added in sequence. After being heated with stirring at 70° C. for14 hours, the solvent was removed in vacuo. The residue was partitionedbetween EtOAc and saturated NaHCO₃ solution (30 mL). The organic layerwas separated and the aqueous phase was extracted with EtOAc (30 mL)twice. The combined organic layers were washed with brine, dried overNa₂SO₄ and concentrated in vacuo to afford 320 mg crude product of[(2R,3R,5S)-5-(1-acetoxy-2-methoxy-ethyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 21f), which was used in next step withoutpurification. MS obsd. (ESI⁻) [(M−H)⁻]: 427.

Preparation of5-amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxy-2-methoxy-ethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of[(2R,3R,5S)-5-(1-acetoxy-2-methoxy-ethyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 21f, prepared above) in methanol (25 mL) was addedK₂CO₃ (272 mg, 2 mmol). After being stirred at room temperature for 12hours, the reaction mixture was adjusted to pH 7-8 by addition of HOAc(240 mg, 4 mmol), concentrated in vacuo and the residue was purified andseparated by preparative HPLC to afford 22.6 mg of Example 21-A and 22.3mg of Example 21-B as white solid.

Example 21-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.92-5.98 (m, 1H),4.92-4.95 (m, 1H), 4.29-4.37 (m, 1H), 3.72-3.79 (m, 1H), 3.42-3.51 (m,2H), 3.38 (s, 3H), 2.56-2.68 (m, 1H), 1.91-2.01 (m, 1H). MS obsd. (ESI⁻)[(M−H)⁻]: 343.

Example 21-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.91-5.96 (m, 1H),4.92-4.95 (m, 1H), 4.20-4.28 (m, 1H), 3.85-3.91 (m, 1H), 3.49-3.56 (m,1H), 3.39-3.45 (m, 1H), 3.37 (s, 3H), 2.63-2.73 (m, 1H), 1.95-2.03 (m,1H). MS obsd. (ESI⁻) [(M−H)⁻]: 343.

Example 225-Amino-3-[(2R,3R,5S)-5-(1-hydroxypropyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared according to the following scheme:

Preparation of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylacetate

To a solution of[(2R,3R,5S)-5-(1-acetoxypropyl)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-cl]pyrimidin-3-yl)tetrahydrofuran-3-yl]acetate (compound 1g, 7.0 g, 16.9 mmol) in methanol (200 mL) was addedK₂CO₃ (1.18 g, 8.5 mmol). After being stirred at room temperature for 12hours, the reaction mixture was adjusted to pH 6.0 by addition of HOAc(1.2 g, 17 mmol), concentrated in vacuo and the residue was purified bycolumn chromatography on silica gel (eluting with 1:2 EtOAc in petroleumether) to afford 2.8 g of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylacetate (compound 22a) as a yellow solid. MS obsd. (ESI⁻) [(M−H)⁻]: 369.

Preparation of1-[(2S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-oxo-tetrahydrofuran-2-yl]propylacetate

To a stirred solution of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylacetate (compound 22a, 2.8 g, 7.6 mmol) in THF (100 mL) was addedDess-Martine periodinane (4.8 g, 11.3 mmol). After being stirred at roomtemperature for 2 hours, the resulting solution was filtered and thefiltrate was concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 1:10 methanol in DCM) toafford 2.8 g crude product of1-[(2S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-oxo-tetrahydrofuran-2-yl]propylacetate (compound 22b). MS obsd. (ESI⁻) [M−H)⁻]: 367.

Preparation of1-[(2S,4S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylacetate

To a stirred solution of1-[(2S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-oxo-tetrahydrofuran-2-yl]propylacetate (compound 22b, 2.8 g, 7.6 mmol) in THF (50 mL) was added lithiumtri-tert-butoxyaluminum hydride (1M in THF, 15 mL, 15 mmol). After beingstirred at room temperature for 2 hours, the resulting solution wasquenched by saturated NH₄Cl solution and filtered. The filtrate wasconcentrated in vacuo and the residue was purified by columnchromatography on silica gel (eluting with 1:10 methanol in DCM) toafford 1.76 g crude product of1-[(2S,4S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylacetate (compound 22c). MS obsd. (ESI⁻)[(M−H)⁻]: 369. (Refer toTetrahedron 1984, 40, 125-135).

Preparation of1-[(2S,4S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]propylacetate

To a stirred solution of1-[(2S,4S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylacetate (compound 22c, 1.76 g, 0.46 mmol) in DCM (30 mL) was addedpyridine (154 mg, 1.9 mmol) and trifluoromethanesulfonic anhydride (197mg, 0.7 mmol). After being stirred at room temperature for 2 hours, theresulting solution was washed with water, brine, dried over Na₂SO₄. Theorganic layer was concentrated in vacuo and the residue was purified bycolumn chromatography on silica gel (eluting with 1:10 EtOAc inpetroleum ether) to afford 420 mg of1-[(2S,4S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]propylacetate (compound 22d). MS obsd. (ESI⁻) [(M−H)⁻]: 502.

Preparation of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-methylsulfanyl-tetrahydrofuran-2-yl]propylacetate

To a stirred solution of1-[(2S,4S,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]propylacetate (compound 22d, 420 mg, 0.83 mmol) in DMF (7 mL) was added sodiumthiomethoxide (84 mg, 1.2 mmol). After being stirred at room temperaturefor 2 hours, the resulting solution was diluted with EtOAc, washed withbrine, dried over Na₂SO₄ and concentrated in vacuo to afford crudeproduct of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazol[4,5-d]pyrimidin-3-yl)-4-methylsulfanyl-tetrahydrofuran-2-yl]propylacetate (compound 22e), which was used in next step without furtherpurification. MS obsd. (ESI⁻) [(M−H)⁻]: 399.

Preparation of5-amino-3-[(2R,3R,5S)-5-(1-hydroxypropyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-methylsulfanyl-tetrahydrofuran-2-yl]propylacetate (compound 22e, 200 mg, 0.5 mmol) in methanol (25 mL) was addedK₂CO₃ (272 mg, 2 mmol). After being stirred at room temperature for 12hours, the reaction mixture was adjusted to pH 7.0 by addition of HOAc(120 mg, 2 mmol), concentrated in vacuo and the residue was purified andseparated by preparative HPLC to afford 4.7 mg of Example 22-A and 1.8mg of Example 22-B as white solid.

Example 22-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 6.09-6.16 (m, 1H),4.09-4.16 (m, 1H), 3.97-4.06 (m, 1H), 3.47-3.57 (m, 1H), 2.61-2.72 (m,1H), 2.13 (s, 3H), 1.95-2.06 (m, 1H), 1.41-1.61 (m, 2H), 1.01 (t, J=7.2Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 357.

Example 22-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 6.05-6.12 (m, 1H),4.01-4.11 (m, 2H), 3.65-3.74 (m, 1H), 2.67-2.78 (m, 1H), 2.12 (s, 3H),1.98-2.05 (m, 1H), 1.52-1.65 (m, 1H), 1.31-1.47 (m, 1H), 1.01 (t, J=7.2Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 357.

Example 235-Amino-3-[(2R,3R,5S)-3-azido-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 22, by usingsodium azide instead of sodium thiomethoxide. Example 23 was purifiedand separated by preparative HPLC to afford Example 23-A and Example23-B as white solid.

Example 23-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.93-5.99 (m, 1H),4.95-5.00 (m, 1H), 4.06-4.14 (m, 1H), 3.47-3.55 (m, 1H), 2.63-2.75 (m,1H), 2.03-2.12 (m, 1H), 1.51-1.61 (m, 1H), 1.43-1.51 (m, 1H), 1.01 (t,J=7.40 Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 352.

Example 23-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.92-5.97 (m, 1H),4.94-4.98 (m, 1H), 4.02-4.10 (m, 1H), 3.62-3.68 (m, 1H), 2.72-2.80 (m,1H), 2.06-2.15 (m, 1H), 1.53-1.68 (m, 1H), 1.33-1.45 (m, 1H), 1.00 (t,J=7.40 Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 352.

Example 245-Amino-3-[(2R,3R,5S)-3-hydroxy-5-(1-hydroxyallyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 1, by usingethylene magnesium bromide instead of ethyl magnesium. Example 24 waspurified and separated by preparative HPLC to afford Example 24-A andExample 24-B as white solid.

Example 24-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.96-6.01 (m, 1H),5.86-5.96 (m, 1H), 5.33-5.43 (m, 1H), 5.16-5.22 (m, 1H), 4.91-4.95 (m,1H), 4.16-4.23 (m, 1H), 4.09-4.16 (m, 1H), 2.53-2.63 (m, 1H), 1.87-1.95(m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 357.

Example 24-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.93-5.95 (m, 1H),5.84-5.92 (m, 1H), 5.37 (td, J=1.76, 17.32 Hz, 1H), 5.17-5.23 (m, 1H),4.93-4.99 (m, 1H), 4.29 (br. s., 1H), 4.22 (d, J=4.52 Hz, 1H), 2.57-2.68(m, 1H), 1.88-1.98 (m, 1H). MS obsd. (ESI⁻) [(M−H)⁻]: 357.

Example 255-Amino-3-((2R,3R,5S)-3-azido-5-((S)-1-hydroxyethyl)tetrahydrofuran-2-yl)thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione

The title compound was prepared according to the following scheme:

Preparation of(2R,3S,5R)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]tetrahydrofuran-2,3,4-triol

To a stirred solution of D-arabinose (50 g, 0.33 mol) in DMF (500 mL)was added imidazole (45 g, 0.66 mol) and tert-butylchlorodiphenylsilane(109 g, 0.4 mol). After being stirred at room temperature for 2 hours,the resulting solution was diluted with EtOAc (2000 mL), washed withwater, brine and dried over Na₂SO₄. The organic layer was concentratedin vacuo and the residue was purified by column chromatography on silicagel (eluting with 1:11 EtOAc in petroleum ether) to afford 33 g of(2R,3S,5R)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]tetrahydrofuran-2,3,4-triol(compound 25a).

Compound 25a: ¹H NMR (400 MHz, CDCl₃) δ ppm: 7.66-7.75 (m, 4H),7.38-7.52 (m, 6H), 5.44-5.50 (m, 1H), 4.30 (d, J=1.76 Hz, 1H), 4.25 (m,1H), 4.09 (m, 1H), 3.93-4.00 (m, 1H), 3.84-3.89 (m, 1H), 3.74-3.78 (m,1H), 1.02-1.09 (m, 9H).

Preparation of(3aS,5R,6aS)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol

To a stirred solution of (2R,3S,5R)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]tetrahydrofuran-2,3,4-triol (compound 25a, 33g, 85 mmol) in acetone (250 mL) was added 2,2-dimethoxypropane (13.2 g,127 mmol) and p-toluene sulfonic acid (1 g, 5.8 mmol). After beingstirred at 60° C. for 2 hours, the resulting solution was adjusted to pH7.0 by addition of saturated NaHCO₃ solution and concentrated in vacuo.The residue was purified by column chromatography on silica gel (elutingwith 1:11 EtOAc in petroleum ether) to afford 20 g of(3aS,5R,6aS)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol(compound 25b).

Compound 25b: ¹H NMR (400 MHz, CDCl₃) δ ppm: 7.66-7.71 (m, 4H), 7.41 (d,J=7.78 Hz, 6H), 5.87-5.93 (m, 1H), 4.55-4.60 (m, 1H), 4.42-4.49 (m, 1H),4.04-4.10 (m, 1H), 3.80-3.89 (m, 2H), 1.35 (s, 3H), 1.31 (s, 3H), 1.09(s, 9H).

Preparation of[(3aS,5R,6aS)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]oxy-phenoxy-methanethione

To a solution of(3aS,5R,6aS)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol(compound 25b, 23 g, 50 mmol) in DCM (200 mL) was added O-phenylchloromethanethioate (10.3 g, 60 mmol) and pyridine (7.9 g, 100 mmol) inDCM. After being stirred at room temperature overnight, the resultingmixture was washed with brine, dried over Na₂SO₄, and concentrated invacuo. The residue was purified by column chromatography on silica gel(eluting with 0-30% EtOAc in petroleum ether) to afford 20 g of[(3aS,5R,6aS)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]oxy-phenoxy-methanethione(compound 25c). MS obsd. (ESI⁺) [(M+NH₄)⁺]: 582.

Preparation of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methoxy-tert-butyl-diphenyl-silane

To a solution of[(3aS,5R,6aS)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]oxy-phenoxy-methanethione(compound 25c, 17 g, 30 mmol) in toluene (150 mL) was addedtri(trimethylsilyl)silane(16.4 g, 66 mmol) and azodiisobutyronitrile (98mg, 0.6 mmol), the mixture was heated at 130° C. under nitrogen for 3hours. After the reaction was completed, the reaction was concentratedin vacuo and the residue was purified by column chromatography on silicagel (eluting with 1:10 EtOAc in petroleum ether) to afford 11 g of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methoxy-tert-butyl-diphenyl-silane(compound 25d).

Compound 25d: ¹H NMR (400 MHz, CDCl₃) δ ppm: 7.70 (qd, J=1.89, 5.87 Hz,4H), 7.37-7.46 (m, 6H), 5.78-5.83 (m, 1H), 4.72-4.78 (m, 1H), 4.27-4.35(m, 1H), 3.84 (d, J=6.78 Hz, 2H), 2.25-2.33 (m, 1H), 2.13-2.19 (m, 1H),1.35 (s, 3H), 1.30 (s, 3H), 1.08 (s, 9H).

Preparation of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol

To a solution of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methoxy-tert-butyl-diphenyl-silane(compound 25d, 11 g, 26.6 mmol) in THF (100 mL) was added TBAF solution(1M in THF, 6 mL, 6 mmol) with stirring. After being stirred at roomtemperature for 4 hours, the reaction solution was washed with saturatedNH₄Cl solution, dried over Na₂SO₄, concentrated in vacuo and the residuewas purified by column chromatography on silica gel (eluting with 1:2EtOAc in petroleum ether) to afford 5.8 g of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol(compound 25e).

Compound 25e: ¹H NMR (400 MHz, CDCl₃) δ ppm: 5.81-5.89 (m, 1H),4.75-4.83 (m, 1H), 4.32-4.41 (m, 1H), 3.81-3.91 (m, 1H), 3.60-3.70 (m,1H), 2.21-2.28 (m, 1H), 1.97-2.09 (m, 1H), 1.57-1.59 (s, 6H).

Preparation of(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde

To a stirred solution of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol(compound 25e, 2 g, 11.5 mmol) in THF (20 mL) was added Dess-Martineperiodinane (7.2 g, 17.2 mmol). After being stirred room temperature for2 hours, the resulting solution was filtered and the filtrate wasconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (eluting with 1:3 EtOAc in petroleum ether) to afford 1.2g of(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde(compound 25f). MS obsd. (ESI⁺) [(M+NH₄)⁺]: 190.

Preparation of1-[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethanol

To a solution of(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde(compound 25f, 800 mg, 1 mmol) in THF (20 mL) was added methyl magnesiumbromide (2M in THF, 0.7 mL, 1.4 mmol) at −20° C. under argon. Afterbeing stirred at −20° C. for 20 hours, the reaction was quenched bysaturated NH₄Cl solution, extracted with EtOAc (30 mL) three times. Theorganic layers were combined and concentrated in vacuo to afford 400 mgcrude product of1-[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethanol(compound 25g), which was used in next step without furtherpurification. MS obsd. (ESI⁺) [(M+NH₄)⁺]: 206.

Preparation of1-[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethylbenzoate

To a cooled solution of1-[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethanol(compound 25g, 200 mg, 1.06 mmol) in DCM (8 mL) was added benzoylchloride (178 mg, 1.28 mmol) and DMAP (259 mg, 2 mmol). After theaddition, the mixture was warmed naturally to room temperature andstirred at room temperature overnight. The resulting mixture was dilutedwith EtOAc and washed with a saturated aqueous solution of NH₄Cl. Theorganic layer was dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (elutingwith 1:3 EtOAc in petroleum ether) to afford 170 mg of1-[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethylbenzoate (compound 25h).

Compound 25h: ¹H NMR (400 MHz, CDCl₃) δ ppm: 8.05 (s, 2H), 7.56-7.63 (m,1H), 7.46 (s, 2H), 5.85 (d, J=4.02 Hz, 1H), 5.38-5.52 (m, 1H), 4.73-4.83(m, 1H), 4.13-4.25 (m, 1H), 2.22 (d, J=4.77 Hz, 2H), 1.62 (s, 3H), 1.46(d, J=6.27 Hz, 3H), 1.34 (s, 3H).

Preparation of 1-[(2S,4S)-4-hydroxy-5-methoxy-tetrahydrofuran-2-yl]ethylbenzoate

A solution of1-[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]ethylbenzoate (compound 25h, 170 mg, 18 mmol) in the HCl solution (0.1N inMeOH, 3 mL) was stirred at room temperature overnight. The resultingmixture was neutralized by ammonium hydroxide and concentrated in vacuo.The residue was suspended in EtOAc and then filtered, the filtrate wasconcentrated in vacuo to afford 148 mg crude product of1-[(2S,4S)-4-hydroxy-5-methoxy-tetrahydrofuran-2-yl]ethyl benzoate(compound 25i), which was used in the next step directly. MS obsd.(ESI⁺) [(M+H)⁺]: 267.

Preparation of1-[(2S,4S)-5-methoxy-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]ethylbenzoate

To a solution of1-[(2S,4S)-4-hydroxy-5-methoxy-tetrahydrofuran-2-yl]ethyl benzoate(compound 25i, 483 mg) and DMAP (885 mg, 7.3 mmol) in DCM (20 mL) wasadded trifluoromethanesulfonic anhydride (665 mg, 2.36 mmol) at 0° C.After being stirred at room temperature for 0.5 hr, the reaction wasquenched by saturated NaHCO₃ solution and extracted with DCM threetimes. The organic layers were combined, dried over Na₂SO₄ andconcentrated in vacuo to afford 740 mg crude product of1-[(2S,4S)-5-methoxy-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]ethylbenzoate (compound 25j) as an oil, which was used in next step directlywithout further purification. MS obsd. (ESI⁺) [(M+H)⁺]: 400.

Preparation of 1-[(2S,4R)-4-azido-5-methoxy-tetrahydrofuran-2-yl]ethylbenzoate

To a solution of1-[(2S,4S)-5-methoxy-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]ethylbenzoate (compound 25j, 400 mg, 1 mmol) in DMF (2 mL) was added sodiumazide (65 mg, 1.05 mmol) at room temperature and the mixture was stirredat room temperature for 16 hours. The reaction mixture was partitionedbetween EtOAc and H₂O, the organic layer was separated and the aqueouslayer was extracted with EtOAc twice. The organic layers were combined,washed with brine, dried over Na₂SO₄ and concentrated in vacuo to afford600 mg crude product of1-[(2S,4R)-4-azido-5-methoxy-tetrahydrofuran-2-yl]ethyl benzoate(compound 25k), which was used in next step without furtherpurification.

Preparation of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-azido-tetrahydrofuran-2-yl]ethylbenzoate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (186 mg, 1 mmol)in ACN (10 mL) was added BSA (630 mg, 3 mmol). The resulting reactionmixture was then stirred at 70° C. under argon for 0.5 hour to form aclear solution. After the solution was cooled to room temperature,1-[(2S,4R)-4-azido-5-methoxy-tetrahydrofuran-2-yl]ethyl benzoate(compound 25k, 300 mg, 1.0 mmol) and TMSOTf (1.15 g, 5 mmol) were addedin sequence. After being heated at 70° C. for 14 hours, the solvent wasremoved in vacuo. The residue was partitioned between EtOAc andsaturated NaHCO₃ solution (30 mL). The organic layer was separated andthe aqueous phase was extracted with EtOAc (30 mL) twice. The combinedorganic layers were washed with brine, dried over Na₂SO₄ andconcentrated in vacuo to afford 600 mg crude product of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-azido-tetrahydrofuran-2-yl]ethylbenzoate (compound 25I), which was used in next step without furtherpurification. MS obsd. (ESI⁺) [(M+H)⁺]: 442.

Preparation of5-amino-3-[(2R,3R,5S)-3-azido-5-(1-hydroxyethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-azido-tetrahydrofuran-2-yl]ethylbenzoate (compound 25I, 600 mg, crude) in MeOH (8 mL) was added K₂CO₃(187 mg, 1.4 mmol). After being stirred at room temperature for 5 hours,the reaction mixture was concentrated in vacuo and the residue waspurified by preparative HPLC to afford 30 mg of5-amino-3-[(2R,3R,5S)-3-azido-5-(1-hydroxyethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 25) as a white solid.

Example 25: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.26 (s, 1H), 6.96 (br.s., 2H), 5.76 (d, J=3.3 Hz, 1H), 5.03 (dt, J=7.4, 2.8 Hz, 1H), 4.71 (d,J=5.0 Hz, 1H), 3.80 (dt, J=9.2, 6.1 Hz, 1H), 3.57-3.67 (m, 1H),2.53-2.68 (m, 1H), 2.04 (ddd, J=13.2, 6.0, 2.6 Hz, 1H), 1.05 (d, J=6.3Hz, 3H). MS obsd. (ESI⁻) [(M−H)⁻]: 338.

Example 263-[(2R,3R,5S)-3-allyl-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared according to the following scheme:

Preparation of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propylbenzoate

To a stirred solution of crude1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 1e, 8.08 g, 40.0 mmol) and pyridine (16.1 mL, 200 mmol) in DCMwas added benzoyl chloride (5.0 mL, 43.0 mmol) dropwise at 0° C. Afterthe addition, the mixture was warmed to room temperature and stirred atroom temperature overnight. The resulting mixture was washed with 1Nhydrochloric acid, brine, dried over Na₂SO₄ and concentrated in vacuo.The residue was purified by column chromatography on silica gel (elutingwith 1:3 EtOAc in petroleum ether) to afford 6.86 g of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propylbenzoate (compound 26a).

Preparation of 1-[(2S,4R)-4,5-diacetoxytetrahydrofuran-2-yl]propylbenzoate

To a stirred solution of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propylbenzoate (compound 26a, 6.73 g, 22.0 mmol) and acetic anhydride (11 mL)in acetic acid (44 mL) and chloroform (11 mL) was added concentratedsulfuric acid (200 uL) dropwise. After being stirred at room temperatureovernight, the resulted mixture was diluted with EtOAc (100 mL) andwashed with a saturated aqueous solution of NaHCO₃ (100 mL) three times.The combined organic layers were dried over Na₂SO₄ and concentrated invacuo. The residue was purified by column chromatography on silica gel(eluting with 1:20 to 1:10 EtOAc in petroleum ether) to afford 5.1 g1-[(2S,4R)-4,5-diacetoxytetrahydrofuran-2-yl]propyl benzoate (compound26b) as a viscous oil.

Preparation of1-[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propylbenzoate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (2.82 g, 16.8mmol) in ACN (100 mL) was added BSA (10.4 mL, 42 mmol). The resultingreaction mixture was then stirred at 70° C. under argon for 0.5 hour toform a clear solution. After the solution was cooled to roomtemperature, 1-[(2S,4R)-4,5-diacetoxytetrahydrofuran-2-yl]propylbenzoate (compound 26b, 4.9 g, 14.0 mmol) and TMSOTf (4.7 mL, 2.3 21mmol) were added in sequence. After being heated with stirring at 70° C.for 14 hours, the solvent was removed in vacuo. The residue waspartitioned between EtOAc and saturated NaHCO₃ solution (30 mL). Theorganic layer was separated and the aqueous phase was extracted withEtOAc (30 mL) twice. The combined organic layers were washed with brine,dried over Na₂SO₄ and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (eluting with 1:200 methanol in DCM)to afford 5.27 g crude product of1-[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propylbenzoate (compound 26c) as a light yellow solid. MS obsd. (ESI⁺)[(M+H)⁺]: 475.

Preparation of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylbenzoate

To a solution of1-[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propylbenzoate (compound 26c, 4.98 g, 10.5 mmol) in methanol (105 mL) wasadded K₂CO₃ (1.38 g, 10.0 mmol). After being stirred at room temperaturefor 1 hour, the reaction was adjusted to pH 7.0 with HOAc (1.2 g, 20.0mmol), concentrated in vacuo and the residue was purified by columnchromatography on silica gel (eluting with 1:200 methanol in DCM) toafford 4.5 g of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylbenzoate (compound 26d) as a light brown solid. MS obsd. (ESI⁺)[(M+H)⁺]: 433.

Preparation of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-phenoxycarbothioyloxy-tetrahydrofuran-2-yl]propylbenzoate

To a solution of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propylbenzoate (compound 26d, 4.32 g, 10.0 mmol) in DCM (60 mL) was added DMAP(2.44 g, 20 mmol) and O-phenyl chloromethanethioate (1.6 mL, 12.0 mmol)with stirring. After being stirred at room temperature for 2 hours, theresulting solution was concentrated in vacuo and the residue waspurified by column chromatography on silica gel (eluting with 1:10 to1:1 EtOAc in petroleum ether) to afford 1.9 g of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-phenoxycarbothioyloxy-tetrahydrofuran-2-yl]propylbenzoate (compound 26e). MS obsd. (ESI⁺) [(M+H)⁺]: 569.

Preparation of1-[(2S,4R,5R)-4-allyl-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propylbenzoate

A mixture of1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-phenoxycarbothioyloxy-tetrahydrofuran-2-yl]propylbenzoate (compound 26e, 1.14 g, 2.0 mmol), 2,2′-azobisisobutyronitrile(168 mg, 1 mmol) and allyl(tributyl)stannane (3.08 mL, 10 mmol) inanhydrous toluene (15 mL) was degassed with argon and then heated withstirring at 80° C. for 4 hours. The resulting mixture was stirred withsaturated aqueous NH₄F at room temperature for 2 hours and extractedwith DCM twice. The combined organic layers were dried over Na₂SO₄,concentrated in vacuo and the residue was purified by columnchromatography on silica gel (eluting with 1:3 EtOAc in petroleum ether)to afford 820 mg of1-[(2S,4R,5R)-4-allyl-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propylbenzoate (compound 26f) as a brown solid. MS obsd. (ESI⁺) [(M+H)⁺]: 457.

Preparation of3-[(2R,3R,5S)-3-allyl-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of1-[(2S,4R,5R)-4-allyl-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propylbenzoate (compound 26f, 800 mg, 1.75 mmol) in methanol (25 mL) was addedK₂CO₃ (483 mg, 3.5 mmol). After being stirred at room temperature for 12hours, the reaction was diluted by saturated NH₄Cl solution andextracted with DCM. The organic layers were combined and concentrated invacuo. The residue was purified by preparative HPLC to afford 200 mg of3-[(2R,3R,5S)-3-allyl-5-(1-hydroxypropyl)tetrahydrofuran-2-yl]-5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 26). 100 mg of Example 26 was further separated bysupercritical fluid chromatography (SFC) to afford 32.0 mg of Example26-A and 30.8 mg of Example 26-B as white solid.

Example 26: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.32 (br. s., 1H), 6.89(br. s., 2H), 5.64-5.81 (m, 2H), 4.92-5.10 (m, 2H), 4.45-4.63 (m, 1H),3.74-3.93 (m, 1H), 3.37-3.48 (m, 1H), 2.97-3.14 (m, 1H), 2.12-2.39 (m,3H), 1.61-1.79 (m, 1H), 1.36-1.52 (m, 1H), 1.14-1.29 (m, 1H), 0.88 (q,J=7.36 Hz, 3H) MS obsd. (ESI⁺) [(M+H)⁺]: 353.

Example 26-A: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.24 (br. s., 1H), 6.87(br. s., 2H), 5.66-5.80 (m, 2H), 4.95-5.11 (m, 2H), 4.45 (d, J=6.53 Hz,1H), 3.82-3.93 (m, 1H), 3.39-3.49 (m, 1H), 2.95-3.06 (m, 1H), 2.14-2.30(m, 3H), 1.66 (ddd, J=4.89, 7.09, 12.11 Hz, 1H), 1.34-1.49 (m, 1H),1.18-1.33 (m, 1H), 0.88 (t, J=7.28 Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]:353.

Example 26-B: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.28 (br. s., 1H), 6.87(br. s., 2H), 5.65-5.79 (m, 2H), 4.92-5.10 (m, 2H), 4.57 (d, J=4.77 Hz,1H), 3.78 (q, J=6.78 Hz, 1H), 3.39-3.50 (m, 1H), 3.03-3.15 (m, 1H), 2.35(ddd, J=5.77, 8.66, 12.42 Hz, 1H), 2.16 (t, J=7.15 Hz, 2H), 1.73 (td,J=7.34, 12.42 Hz, 1H), 1.42-1.55 (m, 1H), 1.14-1.28 (m, 1H), 0.87 (t,J=7.28 Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]: 353.

Example 27 Example 27-A and Example 27-B:5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-propyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dioneand5-amino-3-[(2R,3R,5S)-5-[(1R)-1-hydroxypropyl]-3-propyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

A solution of Example 26-A (50 mg, 0.15 mmol) in methanol was stirredwith PtO₂ (10 mg) under hydrogen atmosphere for 4 hours at roomtemperature. The reaction mixture was filtered to remove PtO₂. Thefiltrate was concentrated in vacuo and the residue was purified andseparated by preparative HPLC to afford 32.1 mg of Example 27-A as awhite solid.

Example 27-A: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 10.86-11.27 (br. s, 1H),6.81-7.00 (br. s, 2H), 5.64-5.72 (m, 1H), 4.41-4.49 (m, 1H), 3.82-3.91(m, 1H), 2.81-2.95 (m, 1H), 2.16-2.30 (m, 1H), 1.57-1.69 (m, 1H),1.34-1.50 (m, 4H), 1.22-1.34 (m, 4H), 0.79-0.94 (m, 6H). MS obsd. (ESI⁺)[(M+H)⁺]: 355.

Example 27-B was prepared in analogy to Example 27-A, by using Example26-B instead of Example 26-A.

Example 27-B: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.18-11.47 (br. s., 1H),6.79-7.02 (br. s., 2H), 5.61-5.75 (m, 1H), 4.57 (d, J=4.27 Hz, 1H),3.69-3.85 (m, 1H), 3.45 (m, 1H), 2.94-3.08 (m, 1H), 2.35 (m, 1H), 1.71(m, 1H), 1.42-1.55 (m, 1H), 1.31-1.42 (m, 2H), 1.12-1.31 (m, 3H),0.69-0.95 (m, 6H). MS obsd. (ESI⁺) [(M+H)⁺]: 355.

Example 285-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared according to the following scheme:

Preparation of (2S)-5-oxotetrahydrofuran-2-carboxylic acid

To a solution of (S)-2-aminopentanedioic acid (100 g, 680 mmol) in H₂O(500 mL) was added HCl (140 mL, 1.6 mol) and then NaNO₂ (70.4 g, 1.02mol) in H₂O (100 mL) was added slowly at −5° C.-0° C., and the reactionmixture was stirred at room temperature for 24 hours. The solvent wasconcentrated in vacuo below 50° C. The residue was suspended in EtOAc(500 mL) and filtered. The filtrate was dried over Na₂SO₄ andconcentrated in vacuo to afford 56 g crude product of(2S)-5-oxotetrahydrofuran-2-carboxylic acid (compound 28a) as yellowoil, which was used in next step without further purification.

Preparation of (2S)-5-oxotetrahydrofuran-2-carbonyl chloride

To a solution of (2S)-5-oxotetrahydrofuran-2-carboxylic acid (compound28a, 70 g, 539 mmol) and a drop of DMF in anhydrous DCM (500 mL) wasadded oxalyl dichloride (137 g, 1.07 mol) dropwise. The reaction mixturewas stirred at room temperature for 3 hours. The reaction was thenconcentrated in vacuo to afford 70 g crude product of(2S)-5-oxotetrahydrofuran-2-carbonyl chloride (compound 28b), which wasused in next step without further purification.

Preparation of (5S)-5-propanoyltetrahydrofuran-2-one

To a solution of (2S)-5-oxotetrahydrofuran-2-carbonyl chloride (compound28b, 70 g, 473 mmol) in dry THF (400 mL) was added ethylmagnesiumbromide (173 mL, 520 mmol, 3M in THF) slowly at −78° C. under N₂. Afteraddition, the reaction mixture was stirred at −78° C. for another 2hours. The mixture was then quenched with saturated NH₄Cl solution andextracted with EtOAc (500 mL) twice. The combined organic layers wereconcentrated in vacuo and the residue was purified by columnchromatography on silica gel (eluting with 1:7 to 1:3 EtOAc in petroleumether) to afford 35 g of (5S)-5-propanoyltetrahydrofuran-2-one (compound28c) as a light yellow oil.

Preparation of (5S)-5-(1-hydroxypropyl)tetrahydrofuran-2-one

To a solution of (5S)-5-propanoyltetrahydrofuran-2-one (compound 28c, 35g, 246.5 mmol) was added L-selectride (320 mL, 320 mmol, 1 M in THF) at−78° C. under N₂. After addition, the reaction mixture was stirred at−78° C. for 2 hours. The reaction mixture was then quenched with 2N HCl(200 mL) and extracted with EtOAc (400 mL) twice. The combined organiclayers were washed with brine (100 mL), concentrated in vacuo and theresidue was purified by column chromatography on silica gel (elutingwith 1:7 to 1:3 EtOAc in petroleum ether) to afford 20 g of(5S)-5-(1-hydroxypropyl)tetrahydrofuran-2-one (compound 28d) as a yellowoil. (Refer to Eur. J. Med. Chem. 1997, 32, 617-623).

Preparation of(5S)-5-[(1S)-1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-2-oneand(5S)-5-[(1R)-1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-2-one

To a solution of (5S)-5-(1-hydroxypropyl)tetrahydrofuran-2-one (compound28d, 9 g, 62.5 mmol) in DMF (100 mL) was addedtert-butylchlorodiphenylsilane (42.8 g, 156 mmol) and imidazole (10.6 g,156 mmol) under N₂. After being stirred at 50° C. for 12 hours, themixture was diluted with water and extracted with EtOAc. The combinedorganic layers were washed with brine (100 mL) and concentrated invacuo. The residue was purified by column chromatography on silica gel(eluting with 1:20 to 1:3 EtOAc in petroleum ether) to afford 18 g of(5S)-5-[1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-2-one. 10g of the mixture was further purified and separated by SFC to afford 5.6g of(5S)-5-[(1S)-1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-2-one(compound 28e-S) and 2 g of(5S)-5-[(1R)-1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-2-one(compound 28e-R). (Refer to Tetrahedron. 1997, 53, 6281-6294).

Compound 28e-S: ¹H NMR (CDCl₃ 400 MHz) δ ppm: 7.73-7.69 (m, 4H),7.46-7.39 (m, 6H), 4.56 (m, 1H), 3.66 (m, 1H), 2.64-2.47 (m, 2H),2.20-2.15 (m, 1H), 1.72-1.67 (m, 1H), 1.47-1.42 (m, 1H), 1.15-1.05 (m,9H), 0.82-0.73 (t, 3H).

Compound 28e-R: ¹H NMR (CDCl₃ 400 MHz) δ ppm: 7.72-7.69 (m, 4H),7.48-7.39 (m, 6H), 4.54 (m, 1H), 3.92 (m, 1H), 2.60-2.47 (m, 2H),2.38-2.31 (m, 1H), 2.19-2.12 (m, 1H), 1.50-1.41 (m, 1H), 1.05 (s, 9H),0.74-0.72 (t, 3H).

Preparation of(3R,5S)-5-[(1S)-1-[tert-butyl(diphenyl)silyl]oxypropyl]-3-methyl-tetrahydrofuran-2-one

To a solution of(5S)-5-[(1S)-1-[tert-butyl(diphenyl)silyl]oxypropyl]tetrahydrofuran-2-one(Compound 28e-S, 3.0 g, 7.8 mmol) in THF (60 mL) at −78° C. was addedlithium diisopropylamide (2M in THF, 5.9 mL, 11.8 mmol) dropwise. Afteraddition, the reaction was stirred at −78° C. for 1 hour. To the mixturewas added iodomethane (5.5 g, 39 mmol) and the mixture was stirred at−78° C. for another 1 hour. The mixture was quenched with saturatedNH₄Cl solution (40 mL), extracted with EtOAc (100 mL) twice. The organiclayers were combined, washed with brine (50 mL), dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (eluting with 1:20 to 1:10 EtOAc in petroleum ether) toafford 2.7 g of(3R,5S)-5-[(1S)-1-[tert-butyl(diphenyl)silyl]oxypropyl]-3-methyl-tetrahydrofuran-2-one(compound 28f) as a colourless oil. (Refer to Tetrahedron. 1997, 53,6281-6294).

Preparation of(3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-one

A solution of(3R,5S)-5-[(1S)-1-[tert-butyl(diphenyl)silyl]oxypropyl]-3-methyl-tetrahydrofuran-2-one(compound 28f, 2.7 g, 6.8 mmol) in THF (10 mL) was added TBAF (1M inTHF, 13.6 mL, 13.6 mmol) and the mixture was stirred at room temperaturefor 12 hours. Then the mixture was concentrated in vacuo and the residuewas purified by column chromatography on silica gel (eluting with 1:30to 1:20 EtOAc in petroleum ether) to afford 1.02 g of(3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-one(compound 28g) as a colourless oil.

Preparation of[(1S)-1-[(2S,4R)-4-methyl-5-oxo-tetrahydrofuran-2-yl]propyl]benzoate

To a solution of(3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-one(compound 28g, 1.0 g, 6.3 mmol), TEA (3.2 g, 31.2 mmol) and DMAP (100mg) in DCM (50 mL) was added benzoyl chloride (1.8 g, 12.6 mmol) slowlyat 0° C. The mixture was stirred at 25° C. for 4 hours and then quenchedby saturated NaHCO₃ solution, extracted with EtOAc (100 mL) twice. Theorganic layers were combined, washed with brine (50 mL), dried overNa₂SO₄ and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 1:20 to 1:5 EtOAc inpetroleum ether) to afford 1.4 g of[(1S)-1-[(2S,4R)-4-methyl-5-oxo-tetrahydrofuran-2-yl]propyl] benzoate(compound 28h) as a colourless oil.

Preparation of[(1S)-1-[(2S,4R)-5-hydroxy-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate

To a solution of[(1S)-1-[(2S,4R)-4-methyl-5-oxo-tetrahydrofuran-2-yl]propyl] benzoate(compound 28h, 1.3 g, 5.0 mmol) in THF (100 mL) was added diisobutylaluminium hydride (11 mL, 11 mmol) dropwise at −78° C. and the mixturewas stirred at −78° C. for 2 hours. The mixture was quenched bysaturated NH₄Cl solution (5 mL) and extracted with EtOAc (100 mL) twice.The organic layers were combined, washed with brine (50ml), dried overNa₂SO₄ and concentrated in vacuo to afford 1.2 g crude product of[(1S)-1-[(2S,4R)-5-hydroxy-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate (compound 28i), which was used in next step without furtherpurification.

Preparation of[(1S)-1-[(2S,4R)-5-acetoxy-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate

To a solution of[(1S)-1-[(2S,4R)-5-hydroxy-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate (compound 28i, crude, 1.2 g, 4.5 mmol) in pyridine (60 mL) wasadded acetic acid anhydride (0.918 g, 9 mmol) and DMAP (200 mg) withstirring. After being stirred 25° C. for 2 hours, the mixture wasquenched with saturated NaHCO₃ solution and extracted with EtOAc (40mL). The organic layers were combined, washed with brine (50 mL), driedover Na₂SO₄ and concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (eluting with 1:20 to 1:5 EtOAc inpetroleum ether) to afford 1.0 g of[(1S)-1-[(2S,4R)-5-acetoxy-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate (compound 28j) as a colourless oil.

Preparation of[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate(compound 28k-A) and[(1R)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate(compound 28k-B)

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (872 mg, 4.7mmol) in ACN (20 mL) was added BSA (913.5 mg, 4.5 mmol). The resultingreaction mixture was then stirred at 70° C. under argon for 2 hours toform a clear solution. After the solution was cooled to roomtemperature,[(1S)-1-[(2S,4R)-5-acetoxy-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate (compound 28j, 294 mg, 0.95 mmol) and TMSOTf (1.44 g, 6.6 mmol)were added in sequence. After being stirred with stirring at 20° C. for14 hrs, the solvent was removed in vacuo. The residue was partitionedbetween EtOAc and saturated NaHCO₃ solution (30 mL). The organic layerwas separated and the aqueous phase was extracted with EtOAc (30 mL)twice. The combined organic layers were washed with brine, dried overNa₂SO₄ and concentrated in vacuo. The residue was purified and separatedby preparative HPLC to afford 9.7 mg of[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate (compound 28k-A) and 8.4 mg of[(1R)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate (compound 28k-B) as white solid.

Compound 28k-A: ¹H NMR (400 MHz, CD₃OD) δ ppm: 7.97-8.05 (m, 2H),7.55-7.62 (m, 1H), 7.41-7.48 (m, 2H), 5.77 (d, J=4.14 Hz, 1H), 5.19-5.27(m, 1H), 4.35 (dt, J=8.38, 6.48 Hz, 1H), 3.16 (dd, J=7.40, 4.27 Hz, 1H),2.57 (dt, J=12.33, 8.77 Hz, 1H), 1.67-1.87 (m, 3H), 1.16 (d, J=7.28 Hz,3H), 0.95 (t, J=7.47 Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]: 431.

Compound 28k-B: ¹H NMR (400 MHz, CD₃OD) δ ppm: 8.03-8.08 (m, 2H),7.61-7.66 (m, 1H), 7.48-7.55 (m, 2H), 6.41 (d, J=7.65 Hz, 1H), 5.06 (td,J=6.65, 4.27 Hz, 1H), 4.75 (d, J=7.15 Hz, 1H), 2.72-2.84 (m, 1H),2.25-2.42 (m, 1H), 1.97-2.06 (m, 1H), 1.75-1.85 (m, 2H), 0.97 (t, J=7.40Hz, 3H), 0.89 (d, J=6.90 Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]: 431.

Preparation of5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of compound[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-methyl-tetrahydrofuran-2-yl]propyl]benzoate (compound 28k-A, 120 mg, 0.27 mmol) in MeOH (2 mL) was addedK₂CO₃ (58 mg, 0.42 mmol). After being stirred at room temperature for 5hours, the reaction mixture was adjusted to pH 7 by bubbling CO₂ andthen concentrated in vacuo. The residue was purified by preparative HPLCto afford 24 mg of5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxypropyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 28) as a white solid.

Example 28: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.81 (d, J=7.03 Hz, 1H),4.03-4.18 (m, 1H), 3.45-3.48 (m, 1H), 3.05-3.11 (m, 1H), 2.37-2.42 (m,1H), 1.80-1.85 (m, 1H), 1.40-1.62 (m, 2H), 1.11 (d, J=6.78 Hz, 3H), 1.00(t, J=7.40 Hz, 3H). MS obsd. (ESI⁺) [(M+H)⁺]: 327.

Example 295-Amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxybut-2-ynyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 28 by using1-propynyl magnesium bromide instead of ethyl magnesium bromide. Afterbeing purified by preparative HPLC,5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxybut-2-ynyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 29) was afforded as a white solid.

Example 29: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.83-5.85 (m, 1H), 4.74-4.78(m, 1H), 4.42-4.44 (m, 1H), 4.12-4.13 (m, 1H), 3.15-3.17 (m, 1H),1.87-1.89(m, 1H), 1.83-1.84 (m, 3H), 1.10-1.12 (m, 3H). MS obsd. (ESI⁺)[(M+H)⁺]: 337.

Example 305-Amino-3-[(2R,3R,5S)-5-[(S)-cyclopropyl(hydroxy)methyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 28 by usingcyclopropyl magnesium bromide instead of ethyl magnesium bromide. Afterbeing purified by preparative HPLC,5-amino-3-[(2R,3R,5S)-5-[(S)-cyclopropyl(hydroxy)methyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 30) was afforded as a white solid.

Example 30: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.82 (d, J=6.78 Hz, 1H), 4.20(dt, J=7.87, 5.22 Hz, 1H), 3.05 (d, J=7.78 Hz, 1H), 2.92 (dd, J=8.34,4.58 Hz, 1H), 2.38-2.47 (m, 1H), 1.83 (dt, J=12.39, 7.73 Hz, 1H), 1.10(d, J=6.90 Hz, 3H), 0.87-0.98 (m, 1H), 0.46-0.52 (m, 2H), 0.26-0.39 (m,2H). MS obsd. (ESI⁺) [(M+H)⁺]: 339.

Example 315-Amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxyethyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 29 by using methylmagnesium bromide instead of ethyl magnesium bromide. After beingpurified by preparative HPLC,5-amino-3-[(2R,3R,5S)-5-[(1S)-1-hydroxyethyl]-3-methyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 31) was afforded as a white solid.

Example 31: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.81 (d, J=6.52 Hz, 1H),3.90-4.06 (m, 1H), 3.74-3.86 (m, 1H), 2.97-3.16 (m, 1H), 2.25-2.41 (m,1H), 1.68-1.83 (m, 1H), 1.15 (d, J=6.40 Hz, 3H,), 1.10 (d, J=6.90 Hz,3H). MS obsd. (ESI⁺) [(M+H)⁺]: 313.

Example 325-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-pyrrolidin-1-yl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared according to the following scheme:

Preparation of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methylbenzoate

To a cooled solution of crude[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol(compound 25e, 7.6 g, 43.6 mmol) and TEA (8.6 g, 109 mmol) in DCM wasadded benzoyl chloride (9.1 g, 65.5 mmol) dropwise at 0° C. withstirring. After the addition, the mixture was warmed naturally to roomtemperature and stirred at room temperature overnight. The resultingmixture was washed with 1N hydrochloric acid, brine, dried over Na₂SO₄and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 0-30% EtOAc in petroleumether) to afford 5.8 g of1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propylbenzoate (compound 32a).

Compound 32a: ¹H NMR (400 MHz, CDCl₃) δ ppm: 8.06-8.16 (m, 2H),7.54-7.62 (m, 1H), 7.42-7.52 (m, 2H), 5.88 (d, J=3.76 Hz, 1H), 4.81 (d,J=1.00 Hz, 1H), 4.51-4.65 (m, 2H), 4.46 (dd, J=4.64, 10.16 Hz, 1H),2.27-2.34 (m, 1H), 2.19 (s, 1H), 1.63 (s, 3H), 1.36 (s, 3H).

Preparation of [(2S,4S)-4-hydroxy-5-methoxy-tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(3aS,5S,6aS)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methylbenzoate (compound 32a, 5.1 g, 18 mmol) in MeOH (50 mL) was added H₂SO₄(0.5 mL) at room temperature. After being stirred at 80° C. for 0.5hour, the reaction mixture was cooled to room temperature, neutralizedby solid NaHCO₃ and concentrated in vacuo. The residue was re-dissolvedin EtOAc, washed with water twice. The separated organic layer was driedover Na₂SO₄ and concentrated in vacuo to afford 4.1 g crude product of[(2S,4S)-4-hydroxy-5-methoxy-tetrahydrofuran-2-yl]methyl benzoate(compound 32b) as oil, which was used in next step directly. MS obsd.(ESI⁺) [(M+NH₄)⁺]: 270.

Preparation of[(2S,4S)-5-methoxy-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(2S,4S)-4-hydroxy-5-methoxy-tetrahydrofuran-2-yl]methyl benzoate(compound 32b, 4.1 g, 17 mmol), pyridine (4.8 g, 60 mmol) and DMAP (300mg, 2.5 mmol) in DCM (50 mL) was added trifluoromethanesulfonicanhydride (8.5 g, 30 mmol) at −30° C. After being stirred at −30° C.-0°C. for 2 hours, the reaction was quenched by saturated NaHCO₃ solutionand extracted with DCM three times. The combined organic layers weredried over Na₂SO₄ and concentrated to afford 6.6 g crude product of[(2S,4S)-5-methoxy-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]methylbenzoate (compound 32c) as an oil, which was used in next step withoutfurther purification. MS obsd. (ESI⁻) [(M+H)⁺]: 385.

Preparation of[(2S,4R)-5-methoxy-4-pyrrolidin-1-yl-tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(2S,4S)-5-methoxy-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]methylbenzoate (compound 32c, 400 mg, 1.04 mmol) in DMF (3 mL) was addedpyrrolidine (142 mg, 2.0 mmol) at room temperature and the mixture wasstirred at room temperature for 16 hours. The reaction mixture wasdiluted by water and extracted with EtOAc, the organic layers werecombined and concentrated in vacuo to afford 230 mg crude product of[(2S,4R)-5-methoxy-4-pyrrolidin-1-yl-tetrahydrofuran-2-yl]methylbenzoate (compound 32d) as an oil, which was used in next step withoutfurther purification. MS obsd. (ESI⁺) [(M+H)⁺]: 306.

Preparation of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-pyrrolidin-1-yl-tetrahydrofuran-2-yl]methylbenzoate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (138 mg, 0.75mmol) in ACN (5 mL) was added BSA (535 mg, 2.6 mmol). The resultingreaction mixture was then stirred at 70° C. under argon for 0.5 hour toform a clear solution. After the solution was cooled to roomtemperature,[(2S,4R)-5-methoxy-4-pyrrolidin-1-yl-tetrahydrofuran-2-yl]methylbenzoate (compound 32d, 230 mg, 0.75 mmol) and TMSOTf (832 mg, 3.75mmol) were added in sequence. After being heated with stirring at 70° C.for 14 hours, the solvent was removed in vacuo. The residue waspartitioned between EtOAc and saturated NaHCO₃ solution (30 mL). Theorganic layer was separated and the aqueous phase was extracted withEtOAc (30 mL) twice. The combined organic layers were washed with brine,dried over Na₂SO₄ and concentrated in vacuo to afford 250 mg crudeproduct of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-pyrrolidin-1-yl-tetrahydrofuran-2-yl]methylbenzoate (compound 32e) as a yellow solid. MS obsd. (ESI⁺) [(M+H)⁻]:458.

Preparation of5-amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-pyrrolidin-1-yl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of compound[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-pyrrolidin-1-yl-tetrahydrofuran-2-yl]methylbenzoate (compound 32e, 250 mg, 0.54 mmol) in MeOH (8 mL) was addedK₂CO₃ (138 mg, 1.0 mmol). After being stirred at room temperature for 5hours, the reaction mixture was concentrated in vacuo and the residuewas purified by preparative HPLC to afford 10.0 mg of5-amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-pyrrolidin-1-yl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 32) as a white solid.

Example 32: ¹H NMR (400 MHz, CD₃OD) δ ppm: 6.08 (d, J=6.8 Hz, 1H),4.30-4.37 (m, 1H), 3.78-3.89 (m, 2H), 3.54-3.61 (m, 1H), 2.57-2.64 (m,2H), 2.50-2.56 (m, 2H), 2.42-2.49 (m, 1H), 2.20-2.30 (m, 1H), 1.79 (m,4H). MS obsd. (ESI⁻) [(M+H)⁺]: 354.

Example 33N-[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl-]methanesulfonamide

The title compound was prepared according to the following scheme:

Preparation of [(2S,4R,5R)-4-azido-5-methoxy-tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(2S,4S)-5-methoxy-4-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]methylbenzoate (compound 32c, 1.1 g, 2.8 mmol) in DMF (5 mL) was added sodiumazide (372 mg, 5.7 mmol) at room temperature and the mixture was stirredat room temperature for 16 hours. The reaction mixture was diluted withwater and extracted with EtOAc, the organic layers were combined andconcentrated in vacuo to afford 1.1 g crude product of[(2S,4R,5R)-4-azido-5-methoxy-tetrahydrofuran-2-yl]methyl benzoate(compound 33a) as an oil, which was used in next step without furtherpurification.

Preparation of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-azido-tetrahydrofuran-2-yl]methylbenzoate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (730 mg, 3.97mmol) in ACN (15 mL) was added BSA (2.8 mg, 13.8 mmol). The resultingreaction mixture was then stirred at 70° C. under argon for 0.5 hour toform a clear solution. After the solution was cooled to roomtemperature, [(2S,4R,5R)-4-azido-5-methoxy-tetrahydrofuran-2-yl]methylbenzoate (compound 33a, crude, 1.1 g, 3.97 mmol) and TMSOTf (4.4 g, 19.5mmol) were added in sequence. After being heated with stirring at 70° C.for 14 hours, the solvent was removed in vacuo. The residue waspartitioned between EtOAc and saturated NaHCO₃ solution (30 mL). Theorganic layer was separated and the aqueous phase was extracted withEtOAc (30 mL) twice. The combined organic layers were washed with brine,dried over Na₂SO₄ and concentrated in vacuo to afford 500 mg crudeproduct of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-azido-tetrahydrofuran-2-yl]methylbenzoate (compound 33b) as a yellow solid. MS obsd. (ESI⁺) [(M+H)⁻]:430.

Preparation of[(2S,4R,5R)-4-amino-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylbenzoate

To a solution of compound[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-azido-tetrahydrofuran-2-yl]methylbenzoate (compound 33b, 200 mg, 0.466 mmoL) and triphenylphosphine (364mg, 1.39 mmoL) in THF (10 mL) was added water (0.5 mL) at roomtemperature. After being stirred at 80° C. for 1 hour, the reaction wasfiltered and the filtrate was concentrated in vacuo to afford 80 mgcrude product of[(2S,4R,5R)-4-amino-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylbenzoate (compound 33c), which was used in next step directly. MS obsd.(ESI⁺) [(M+H)⁺]: 404.

Preparation of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]dipyrimidin-3-yl)-4-(methanesulfonamido)tetrahydrofuran-2-yl]methylbenzoate

To a solution of compound[(2S,4R,5R)-4-amino-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylbenzoate (compound 33c, crude, 80 mg, 0.198 mmoL) in DCM (10 mL) and THF(2 mL) were added TEA (44 mg, 0.436 mmoL) and methanesulfonyl chloride(27 mg, 0.237 mmol) at 0° C. After being stirred at room temperature for2 hours, the reaction mixture was diluted by DCM, washed with water,brine, dried over Na₂SO₄ and concentrated in vacuo to afford 80 mg crudeproduct of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(methanesulfonamido)tetrahydrofuran-2-yl]methylbenzoate (compound 33d), which was used in next step directly. MS obsd.(ESI⁻) [(M−H)⁻]: 480.

Preparation ofN-[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]methanesulfonamide

To a solution of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(methanesulfonamido)tetrahydrofuran-2-yl]methylbenzoate (compound 33d, crude, 80 mg, 0.54 mmol) in MeOH (5 mL) wasadded K₂CO₃ (80 mg, 0.6 mmol). After being stirred at room temperaturefor 5 hours, the reaction mixture was concentrated in vacuo and theresidue was purified by preparative HPLC to afford 8.0 mg ofN-[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]methanesulfonamide(Example 33) as a white solid.

Example 33: 8 mg, ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.45-11.78 (br. s.,1H), 7.82 (d, J=8.5 Hz, 1H), 6.99 (br. s., 2H), 5.79 (d, J=4.8 Hz, 1H),4.68-4.84 (m, 2H), 4.15 (dt, J=12.7, 6.3 Hz, 1H), 3.46 (m, 2H), 2.89 (s,3H), 2.37-2.44 (m, 1H), 1.90-2.00 (m, 1H). MS obsd. (ESI⁺) [(M+H)⁺]:378.

Example 34N-[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]acetamide

The title compound was prepared in analogy to Example 33 by using acylchloride instead of methyl sulfonyl chloride. After being purified bypreparative HPLC,N-[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]acetamide(Example 34) was afforded as a white solid.

Example 34: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 6.09 (dd, J=9.0, 5.8 Hz,1H), 5.08 (t, J=5.6 Hz, 1H), 3.92-4.01 (m, 1H), 3.66 (dd, J=11.4, 6.1Hz, 1H), 3.50-3.53 (m, 1H), 3.10-3.23 (m, 2H), 1.56-1.61 (m, 1H),1.23-1.38 (m, 1H), 0.93 (t, J=7.3 Hz, 1H). MS obsd. (ESI⁺) [(M+H)⁻]:342.

Example 355-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-morpholino-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 32 by usingmorpholine instead of pyrrolidine. After being purified by preparativeHPLC,5-amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-morpholino-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 35) was afforded as a white solid.

Example 35: ¹H NMR (400 MHz, CD₃OD) δ ppm: 6.17 (d, J=5.3 Hz, 1H), 4.26(tt, J=7.5, 4.8 Hz, 1H), 3.88-3.97 (m, 1H), 3.65-3.75 (m, 6H), 2.52-2.64(m, 4H), 2.37 (ddd, J=13.2, 8.8, 7.4 Hz, 1H), 2.21 (ddd, J=13.1, 7.8,5.0 Hz, 1H). MS obsd. (ESI⁺) [(M+H)⁺]: 370.

Example 365-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-(1-piperidyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 32 by usingpiperidine instead of pyrrolidine. After being purified by preparativeHPLC,5-amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-(1-piperidyptetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 36) was afforded as a white solid.

Example 36: ¹H NMR (400 MHz, CD₃OD) δ ppm: 6.16 (d, J=6.0 Hz, 1H), 4.28(d, J=2.8 Hz, 1H), 4.01 (t, J=4.8 Hz, 1H), 3.74 (dd, J=3.6, 11.6 Hz,1H), 3.63 (dd, J=4.8, 12.0 Hz, 1H), 2.54-2.21 (m, 6H), 1.63-1.48 (m,6H). MS obsd. (ESI⁺) [(M+H)⁺]: 368.

Example 375-Amino-3-[(2R,3R,5S)-3-(dimethylamino)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 32 by usingdimethylamine instead of pyrrolidine. After being purified bypreparative HPLC,5-amino-3-[(2R,3R,5S)-3-(dimethylamino)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 37) was afforded as a white solid.

Example 37: ¹H NMR (400 MHz, CD₃OD) δ ppm: 6.08 (d, J=6.52 Hz, 1H),4.26-4.32 (m, 1H), 3.87-3.95 (m, 1H), 3.77 (dd, J=11.92, 2.89 Hz, 1H),3.57 (dd, J=11.92, 3.64 Hz, 1H), 2.41 (ddd, J=13.11, 8.47, 5.02 Hz, 1H),2.27 (s, 6H), 2.19 (ddd, J=12.89, 8.63, 7.59 Hz, 1H). MS obsd. (ESI⁺)[(M+H)⁺]: 328.

Example 38(2R,3S,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carbonitrile

The title compound was prepared according to the following scheme:

Preparation of[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylbenzoate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (4.42 g, 24.0mmol) in ACN (20 mL) was added BSA (14.8 mL, 60.0 mmol). The resultingreaction mixture was then stirred at 70° C. under argon for 0.5 hr toform a clear solution. After the solution was cooled to roomtemperature, [(2S,4R)-4,5-diacetoxytetrahydrofuran-2-yl]methyl benzoate(CAS #: 4613-71-2, Cat. #: MD04725, commercially available fromCarbosynth Limited, 6.45 g, 20.0 mmol) and TMSOTf (5.5 mL, 30.0 mmol)were added in sequence. After being heated with stirring at 70° C. for14 hours, the solvent was removed in vacuo. The residue was partitionedbetween EtOAc and saturated NaHCO₃ solution (30 mL). The organic layerwas separated and the aqueous phase was extracted with EtOAc (100 mL)twice. The combined organic layers were washed with brine, dried overNa₂SO₄ and concentrated in vacuo. The residue was purified by columnchromatography on silica gel to afford 5.7 g of[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylbenzoate (compound 38a) as a light yellow solid.

Preparation of5-amino-3-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38b) and[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]methylbenzoate (compound 38c)

A mixture of[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]methylbenzoate (compound 38a, 4.72 g,10.6 mmol) and potassium carbonate (1.46g, 10.6 mmol) in methanol (106 mL) was stirred at room temperature for3.5 hours. The reaction was quenched by addition of acetic acid (1. 5mL). The resulting mixture was concentrated in vacuo to remove thesolvent and the residue was purified by column chromatography on silicagel (eluting with 0-5% methanol in DCM) to afford 1.0 g of5-amino-3-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38b) and 2.8 g of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]methylbenzoate (compound 38c) as a pale solid.

Preparation of5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of5-amino-3-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38b, 3.8 g, 12.7 mmol) in DMF (30 mL) was added imidazole (2.6g, 38 mmol) and tert-butylchlorodimethylsilane (4.2 g, 28 mmol) withstirring. After being stirred at room temperature for 2 hours, theresulting solution was diluted by EtOAc (200 mL), washed with water,brine, dried over Na₂SO₄. The organic layer was concentrated in vacuoand the residue was purified by column chromatography on silica gel(eluting with 1:5 EtOAc in petroleum ether) to afford 3.3 g of5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38d).

Compound 38d: ¹H NMR (400 MHz, CDCl₃) δ ppm: 11.21 (s, 1H), 6.94 (br.s., 2H), 5.70-5.80 (m, 1H), 5.44 (d, J=4.5 Hz, 1H), 4.79 (ddt, J=6.6,4.4, 2.2 Hz, 1H), 4.11-4.24 (m, 1H), 3.58-3.70 (m, 2H), 2.32 (ddd,J=12.7, 9.4, 6.8 Hz, 1H), 1.80 (ddd, J=12.7, 6.1, 2.0 Hz, 1H), 0.79-0.92(s, 9H), 0 (s, 6H).

Preparation of5-amino-3-[(2R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-oxo-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a solution of5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38d, 3.3 g, 7.8 mmol) in THF (100 mL) was added Dess-Martineperiodinane (3.68 g, 8.76 mmol) with stirring. After being stirred atroom temperature for 2 hours, the resulting solution was filtered andthe filtrate was concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (eluting with 1:10 methanol in DCM)to afford 2.4 g crude product of5-amino-3-[(2R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-oxo-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38e). MS obsd. (ESI⁺) [(M+H)⁺]: 413.

Preparation of5-amino-3-[(2R,3S,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

To a stirred solution of5-amino-3-[(2R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-oxo-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38e, 1.0 g, 2.43 mmol) in THF (5 mL) was added lithiumtri-tert-butoxyaluminum hydride solution (1M in THF, 2.7 mL, 2.7 mmol).After being stirred at room temperature for 2 hours, the resultingsolution was quenched by saturated NH₄Cl solution and filtered. Thefiltrate was concentrated in vacuo and the residue was purified bycolumn chromatography on silica gel (eluting with 1:10 methanol in DCM)to afford 750 mg crude product of5-amino-3-[(2R,3S,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38f). (Refer to Tetrahedron 1984, 40, 125-135).

Compound 38f: ¹NMR (400 MHz, d₆-DMSO) δ ppm: 7.00 (br. s., 2H), 6.08 (d,J=7.5 Hz, 1H), 5.35 (d, J=5.5 Hz, 1H), 4.53-4.65 (m, 1H), 3.91-3.99 (m,1H), 3.82-3.89 (m, 1H), 3.70 (dd, J=10.5, 4.3 Hz, 1H), 2.11 (t, J=8.7Hz, 2H), 0.84-0.86 (m, 9H), 0.84-0.88 (m, 10H), 0.84-0.86 (m, 9H), 0.01(d, J=2.3 Hz, 6H).

Preparation of[5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]-2-oxo-thiazolo[4,5-d]pyrimidin-7-yl]trifluoromethanesulfonate

To a stirred solution of5-amino-3-[(2R,3S,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-hydroxy-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 38f, 100 mg, 0.24 mmol) in DCM (15 mL) was added DMAP (147 mg,1.2 mmol) and trifluoromathanesulfonyl chloride (122 mg, 0.7 mmol).After being stirred at room temperature for 2 hours, the resultingsolution was washed with water, brine, dried over Na₂SO₄. The organiclayer was concentrated in vacuo to afford 120 mg crude product of[5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]-2-oxo-thiazolo[4,5-d]pyrimidin-7-yl]trifluoromethanesulfonate (compound 38g), which was used in next stepwithout further purification. MS obsd. (ESI⁻) [(M−H)⁻]: 679.

Preparation of(2R,3S,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-3-carbonitrile

To a stirred solution of[5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-(trifluoromethylsulfonyloxy)tetrahydrofuran-2-yl]-2-oxo-thiazolo[4,5-d]pyrimidin-7-yl]trifluoromethanesulfonate (compound 38g, crude, 120 mg, 0.2 mmol) in DMF(2 mL) was added sodium cyanide (100 mg, 2.3 mmol). After being stirredat room temperature for 2 hours, the resulting solution was diluted byEtOAc, washed with brine, dried over Na₂SO₄ and concentrated in vacuo toafford 100 mg crude product of(2R,3S,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-3-carbonitrile(compound 38h), which was used in next step without furtherpurification. MS obsd. (ESI⁻) [(M+H)⁺]: 424.

Preparation of(2R,3S,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carbonitrile

To a stirred solution of(2R,3S,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-3-carbonitrile(compound 38h, crude, 100 mg) in THF (5 mL) was added TBAF solution (1Min THF, 6 mL, 6 mmol) at 0° C. After being stirred at room temperaturefor 4 hours, the reaction solution was washed with saturated NH₄Clsolution, dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by preparative HPLC to afford 9 mg of(2R,3S,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)tetrahydrofuran-3-carbonitrile(Example 38) as a white solid.

Example 38: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.11-11.99 (br. s., 1H),7.10 (br. s., 2H), 6.15 (d, J=4.8 Hz, 1H), 4.86 (br. s., 1H), 4.33 (dt,J=9.5, 4.7 Hz, 1H), 4.07-4.20 (m, 1H), 3.50 (d, J=4.8 Hz, 2H), 2.44-2.48(m, 1H), 2.23-2.36 (m, 1H). MS obsd. (ESI⁺) [(M+H)⁻]: 310.

Example 395-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared in analogy to Example 38 by using sodiummethylsulfide instead of sodium cyanide. After being purified bypreparative HPLC,5-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-methylsulfanyl-tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 39) was afforded as a white solid.

Example 39: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.37-11.55 (br. s., 1H),6.91-7.10 (br. s., 2H), 5.94 (d, J=4.8 Hz, 1H), 4.75 (t, J=5.9 Hz, 1H),4.03-4.15 (m, 2H), 3.45-3.55 (m, 3H), 2.09 (s, 3H), 1.92 (ddd, J=12.8,6.8, 4.8 Hz, 1H). MS obsd. (ESI⁺) [(M+H)⁺]: 331

Example 405-Amino-3-[(2R,3R,5S)-3-(1-fluoro-1-methyl-ethyl)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]dipyrimidine-2,7-dione

The title compound was prepared according to the following scheme:

Preparation of (2S)-2-(hydroxymethyl)-2H-furan-5-one

To a solution of ethyl(Z)-3-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]prop-2-enoate (CAS #:91926-90-8, Cat. #: PB1131897,commercially available from Pharma Block(Nanjing) R&D Co., Ltd, 4.0 g, 20.0 mmol) in methanol was addedcatalytic amount of concentrated sulfuric acid (25 μL of 10%concentrated sulfuric acid in methanol). The mixture was stirred at roomtemperature for 2 hours. The resulting mixture was concentrated in vacuoand the residue was purified by column chromatography on silica gel(eluting with 0-10% methanol in DCM) to afford 2.25 g of(2S)-2-(hydroxymethyl)-2H-furan-5-one (compound 40b) as a viscous oil.

Preparation of(2S)-2-[[tert-butyl(diphenyl)silyl]oxymethyl]-2H-furan-5-one

To a solution of 2.25 g of (2S)-2-(hydroxymethyl)-2H-furan-5-one(compound 40b, 2.11 g, 16.0 mmol) and imidazole (1.63 g, 24.0 mmol) inDCM was added tert-butyl(chloro)diphenylsilane (5.2 mL, 20.0 mmol)dropwise. The resulting mixture was stirred at room temperature for 2hours. The resulting mixture was washed with brine. The aqueous layerwas extracted with DCM. The organic layers were combined, washed with 1Nhydrochloric acid, dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (elutingwith 0-30% EtOAc in petroleum ether) to afford 4.6 g of(2S)-2-[[tert-butyl(diphenyl)silyl]oxymethyl]-2H-furan-5-one (compound40c) as a white solid. MS obsd. (ESI⁺) [(M+H)⁺]: 353.

Preparation of(5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]tetrahydrofuran-2-one

A solution of(2S)-2-[[tert-butyl(diphenyl)silyl]oxymethyl]-2H-furan-5-one (compound40c, 2.8 g, 8.0 mmol) in EtOAc (40 mL) was stirred with 10% palladium oncarbon (280 mg) under hydrogen atmosphere overnight. The resultingmixture was filtered and the filtrate was concentrated in vacuo toafford 2.7 g of(5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]tetrahydrofuran-2-one(compound 40d) as a viscous oil. MS obsd. (ESI⁺) [(M+H)⁺]: 355.

Preparation of(3S,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-hydroxy-1-methyl-ethyl)tetrahydrofuran-2-one

To a cooled solution of(5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]tetrahydrofuran-2-one(compound 40d, 5.00 g, 14.0 mmol) in dry tetrahydrofuran (28 mL) wasadded a solution of lithium bis(trimethylsilyl)azanide (1.3 M in THF,11.8 mL, 15.4 mmol) dropwise at −78° C. under argon. After addition, themixture was stirred at −78° C. for 1 hour. Then distilled acetone (1.23mL, 15.4 mmol) was added dropwise to the mixture and the resultingmixture was stirred at −78° C. for another 2 hours. The reaction wasquenched by saturated NH₄Cl solution and extracted with EtOAc (30 mL)three times. The combined organic layers were dried over Na₂SO₄ andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (eluting with 0-30% EtOAc in petroleum ether) to afford5.7 g of(3S,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-hydroxy-1-methyl-ethyl)tetrahydrofuran-2-one(compound 40e) as a light yellow oil. MS obsd. (ESI⁺) [(M+NH₄)⁺]: 430.(For the synthesis, please refer to: Tetrahedron 1997, 53, 6281-6294).

Preparation of(3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-one

To a solution of diethylaminosulfur trifluoride (414 μL, 3.0 mmol) inDCM (10 mL) at −78° C. was added a solution of (3S,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-hydroxy-1-methyl-ethyl)tetrahydrofuran-2-one(compound 40e, 1.03 g, 2.5 mmol) in DCM (10 mL) dropwise. The resultingmixture was warmed up to room temperature and stirred at roomtemperature overnight. The resulting mixture was concentrated in vacuoand the residue was purified by column chromatography on silica gel(eluting with 0-20% EtOAc in petroleum ether) to afford 820 mg of(3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-one(compound 40f) as a viscous oil. MS obsd. (ESI⁺) [(M+H)⁺]: 415.

Preparation of[(3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-yl]acetate

To a cooled solution of(3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-one(compound 40f, 820 mg, 1.92 mmol) in dry DCM (10 mL) at −78° C. wasadded diisobutyl aluminium hydride (1.0 M in toluene, 6.0 mL, 6.0 mmol)dropwise. The resulting mixture was stirred at −78° C. for 1 hour. Thento the mixture was added pyridine (790 mg, 10 mmol), acetic anhydride(0.93 mL, 10.0 mmol) and DMAP (732 mg, 6.0 mmol). The reaction mixturewas allowed to warm to −20° C. slowly and stirred at −20° C. for severalhours until the reaction was complete. The resulted mixture was quenchedby brine and extracted with EtOAc (30 mL) three times. The organiclayers were combined, dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by column chromatography on silica gel (elutingwith 0-20% EtOAc in petroleum ether) to afford 360 mg of[(3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-yl]acetate (compound 40g) as a viscous oil. MS obsd. (ESI⁺) [(M+H)⁺]: 459.

Preparation of5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

A mixture of 5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (162mg, 0.87 mmol) and bis(trimethylsilyl)acetamide (527 mg, 2.61 mmol) washeated with stirring at 75° C. under argon until the mixture becameclear. The mixture was cooled to room temperature. To the previousreaction mixture,[(3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-yl]acetate (compound 40g, 280 mg, 1.02 mmol) andtrimethylsilyltrifluoromethanesulfonate (290 mg, 1.31 mmol) wereintroduced. The resulting mixture was heated at 75° C. under argon for 3hours. Then the resulting mixture was concentrated in vacuo to removethe solvent and the residue was purified by column chromatography onsilica gel (eluting with 0-5% methanol in DCM) to afford 225 mg of5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyptetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 40h) as a brown solid. MS obsd. (ESI⁻) [(M+H)⁺]: 583.

Preparation of5-amino-3-[(2R,3R,5S)-3-(1-fluoro-1-methyl-ethyl)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

A mixture of5-amino-3-[(2R,3R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-3-(1-fluoro-1-methyl-ethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(compound 40h, 66 mg, 0.12 mmol) and NH₄F (133 mg, 3.6 mmol) in methanolwas heated under reflux for 1.5 hrs. The resulting mixture wasconcentrated in vacuo and the residue was purified by preparative HPLCto afford 10 mg of5-amino-3-[(2R,3R,5S)-3-(1-fluoro-1-methyl-ethyl)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 40) as a white solid.

Example 40: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.27-11.40 (br. s., 1H),6.90-7.08 (br. s., 2H), 6.03-6.10 (m, 1H), 4.65-4.74 (m, 1H), 3.92-4.03(m, 1H), 3.42-3.53 (m, 2H), 3.18-3.30 (m, 1H), 2.10-2.23 (m, 1H),1.96-2.06 (m, 1H), 1.19-1.42 (m, 6H). MS obsd. (ESI⁺) [(M+H)⁺]: 345.

Example 415-Amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-(2-methylallyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

The title compound was prepared according to the following scheme.

Preparation of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-phenoxycarbothioyloxy-tetrahydrofuran-2-yl]methylbenzoate

A mixture of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]methylbenzoate (compound 38c, 2.08 g, 5.0 mmol), O-phenyl chloromethanethioate(0.80 mL) and DMAP (1.22 g, 10.0 mmol) in DCM (50 mL) was stirred atroom temperature overnight. The resulting mixture was washed with brine,dried over Na₂SO₄, and concentrated in vacuo. The residue was purifiedby column chromatography on silica gel (eluting with 0-30% EtOAc inpetroleum ether) to afford 2.20 g of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-phenoxycarbothioyloxy-tetrahydrofuran-2-yl]methylbenzoate (compound 41a) as a pale solid. MS obsd. (ESI⁻) [(M−H)⁻]: 539.

Preparation of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(2-methylallyl)tetrahydrofuran-2-yl]methylbenzoate

A mixture of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-phenoxycarbothioyloxy-tetrahydrofuran-2-yl]methylbenzoate (compound 41a, 324 mg, 0.60 mmol),2,2′-azobis(2-methylpropionitrile) (50 mg, 0.30 mmol) andtributyl(2-methylallyl)stannane (0.70 mL, 3.0 mmol) in anhydrous toluene(10 mL) was degassed with argon and then heated with stirring at 80° C.for 4 hours. The resulting mixture was stirred with saturated aqueousNH₄F at room temperature for 2 hours, and then extracted with DCM twice.The combined organic layers were dried over Na₂SO₄ and concentrated invacuo. The residue was purified by column chromatography on silica gel(eluting with 0-30% EtOAc in petroleum ether) to afford 190 mg of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(2-methylallyl)tetrahydrofuran-2-yl]methylbenzoate (compound 41b) as a brown solid. MS obsd. (ESI⁻) [(M−H)⁻]: 441.

Preparation of5-amino-3-[(2R,3R,5S)-5-(hydroxymethyl)-3-(2-methylallyl)tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione

A solution of[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-(2-methylallyl)tetrahydrofuran-2-yl]methylbenzoate (compound 41b, 180 mg, 0.41 mmol) in methanol was stirred withK₂CO₃ (150 mg, 1.09 mmol) at room temperature for 4 hours. The reactionwas quenched by addition of acetic acid and the resulting mixture wasconcentrated in vacuo. The residue was purified by preparative HPLC toafford 41 mg of3-[(2R,3R,5S)-3-allyl-5-(hydroxymethyl)tetrahydrofuran-2-yl]-5-amino-6H-thiazolo[4,5-d]pyrimidine-2,7-dione(Example 41) as a white powder.

Example 41: ¹H NMR (400 MHz, d₆-DMSO) δ ppm: 11.03-11.29 (m, 1H),6.76-7.04 (m, 2H), 5.68 (d, J=6.02 Hz, 1H), 4.62-4.70 (m, 3H), 3.96-4.10(m, 1H), 3.47 (t, J=5.27 Hz, 2H), 3.24-3.30 (m, 1H), 2.16-2.25 (m, 1H),2.13 (d, J=7.53 Hz, 2H), 1.67-1.76 (m, 1H), 1.62 (s, 3H). MS obsd.(ESI⁺) [(M+H)⁺]: 339.

Example 42[(1S)-1-[(2S,4R,5R)-5-(5-Amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]2-methylpropanoate

The title compound was prepared according to the following Scheme.

Preparation of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]2-methylpropanoate

To a cooled solution of(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propan-1-ol(compound 6e, 505.6 mg, 2.5 mmol) in pyridine was added isobutyrylchloride (0.39 mL, 3.72 mmol) dropwise while cooled with an ice-waterbath. After the addition, the mixture was warmed to room temperature andstirred at room temperature overnight.

The resulting mixture was diluted with EtOAc and washed with a saturatedNH₄Cl solution. The organic layer was dried over Na₂SO₄ and concentratedin vacuo. The residue was purified by column chromatography on silicagel (eluting with 0-30% EtOAc in petroleum ether) to afford 470 mg of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]2-methylpropanoate (compound 42a).

Preparation of[(1S)-1-[(2S,4R,5R)-4,5-diacetoxytetrahydrofuran-2-yl]propyl]2-methylpropanoate

To a stirred solution of[(1S)-1-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]propyl]2-methylpropanoate (compound 42a, 470 mg, 1.73 mmol), acetic anhydride(0.81 mL, 8.64 mmol) and acetic acid (0.51 mL, 8.64 mmol) in DCM (10 mL)was added concentrated sulfuric acid (18.4 μL, 0.17 mmol). The resultingmixture was stirred at room temperature overnight. The resulting mixturewas concentrated in vacuo. The residue was purified by columnchromatography on silica gel (eluting with 0-20% EtOAc in petroleumether) to afford 105 mg of[(1S)-1-[(2S,4R,5R)-4,5-diacetoxytetrahydrofuran-2-yl]propyl]2-methylpropanoate (compound 42b).

Preparation of[(1S)-1-[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propyl]2-methylpropanoate

To a suspension of5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (63 mg, 0.34mmol) in ACN (5 mL) was added BSA (252 μL, 1.02 mmol). The resultingreaction mixture was then stirred at 70° C. under argon for 0.5 hour toform a clear solution. After the solution was cooled to roomtemperature,[(1S)-1-[(2S,4R,5R)-4,5-diacetoxytetrahydrofuran-2-yl]propyl]2-methylpropanoate (compound 42b, 105 mg, 0.34 mmol) and TMSOTf (113 μL,0.51 mmol) were added in sequence. After being heated with stirring at70° C. for 14 hours, the resulting mixture was concentrated in vacuo toremove the solvent and the residue was purified by column chromatographyon silica gel (eluting with 0-5% methanol in DCM) to afford 75 mg of[(1S)-1-[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propyl]2-methylpropanoate (compound 42c) as a light yellow solid.

Preparation of[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]2-methylpropanoate

A mixture of[(1S)-1-[(2S,4R,5R)-4-acetoxy-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-2-yl]propyl]2-methylpropanoate (compound 42c, 70 mg,0.16 mmol) and K₂CO₃ (13.2 mg,0.096 mmol) in methanol (0.5 mL) and tetrahydrofuran (2 mL) was stirredat room temperature overnight. The reaction was quenched by addition ofacetic acid (1 drop). The resulting mixture was concentrated in vacuo toremove the solvents and the residue was purified by preparative HPLC toafford 18.2 mg of[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]2-methylpropanoate (Example 42) as a pale solid.

Example 42: ¹H NMR (400 MHz, CD₃OD) δ ppm: 5.89-5.95 (m, 1H), 4.93-5.01(m, 2H), 4.29-4.38 (m, 1H), 2.51-2.63 (m, 2H), 1.83-1.93 (m, 1H),1.58-1.76 (m, 2H), 1.15 (dd, J=4.02, 7.03 Hz, 6H), 0.88-0.95 (m, 3H). MSobsd. (ESI⁺) [(M+H)⁺]: 399.

Example 43 HEK293-Blue-hTLR-7 Cells Assay:

A stable HEK293-Blue-hTLR-7 cell line was purchased from InvivoGen (Cat.#: hkb-ht1r7, San Diego, Calif., USA). These cells were designed forstudying the stimulation of human TLR7 by monitoring the activation ofNF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter genewas placed under the control of the IFN-β␣minimal promoter fused to fiveNF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κBand AP-1 via stimulating HEK-Blue hTLR7 cells with TLR7 ligands.Therefore the reporter expression was regulated by the NF-κB promoterupon stimulation of human TLR7 for 20 hours. The cell culturesupernatant SEAP reporter activity was determined using QUANTI-Blue™ kit(Cat. #: rep-qb1, Invivogen, San Diego, Calif., USA) at a wavelength of640 nm, a detection medium that turns purple or blue in the presence ofalkaline phosphatase.

HEK293-Blue-hTLR7 cells were incubated at a density of 250,000˜450,000cells/mL in a volume of 180 μL in a 96-well plate in Dulbecco's ModifiedEagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v)heat-inactivated fetal bovine serum for 24 h. Then theHEK293-Blue-hTLR-7 cells were incubated with addition of 20 μL testcompound in a serial dilution in the presence of final DMSO at 1% andperform incubation under 37° C. in a CO₂ incubator for 20 hours. Then 20μL of the supernatant from each well was incubated with 180 μLQuanti-blue substrate solution at 37° C. for 2 hours and the absorbancewas read at 620˜655 nm using a spectrophotometer. The signalling pathwaythat TLR7 activation leads to downstream NF-κB activation has beenwidely accepted, and therefore similar reporter assay was also widelyused for evaluating TLR7 agonist (Tsuneyasu Kaisho and Takashi Tanaka,Trends in Immunology, Volume 29, Issue 7, July 2008, Pages 329.sci;Hiroaki Hemmi et al, Nature Immunology 3, 196-200 (2002).

The TLR7 agonism activity in HEK293-hTLR-7 assay of compounds of presentinvention is listed in Table 1. The Examples were tested in the aboveassay and found to have EC50 of about 10 μM to about 90 μM.

TABLE 1 Activity of Compounds in HEK293-hTLR-7 assay Example No.HEK293-hTLR-7 EC₅₀ (μm) ANA-122 446  1-A 52  5-A 48  8-A 87 10-A 63 14-A48 18-A 12 19-A 38 22-A 14 23-A 26 24-A 70 26 29 26-A 12 27-A 15 28 1029 29 30 15 31 70 32 67 39 62 42 51

A reference compound disclosed in patent WO2006066080(A1) as compound122 (herein referred as ANA-122) was also tested for TLR7 agonismactivity in HEK293-hTLR-7 assay mentioned above, EC50 of ANA-122 wasfound to be 446μM.

Example 44 Metabolism of Prodrugs: Formula (II) or Formula (IIa)

A study was undertaken to evaluate the metabolic conversion of prodrugs,formula (II) or formula (IIa), to compounds of formula (I) or formula(Ia) of the present invention. The produgs, formula (II) or formula(IIa), can be metabolized to the active compound of formula (I) orformula (Ia) and other compounds of the invention in the body if theyare served as prodrugs. Hepatocytes are often used to assess the degreeof metabolic conversion of prodrugs in the body of animal or human.

A study was undertaken to evaluate the metabolic conversion of prodrugs,Example 2-A, Example 3-A, Example 4-A, Example 16-A and Example 17-A, tothe corresponding active forms, Example 1-A and Example 14-A, in thepresence of human hepatocytes. The formation of active forms, Example1-A and Example 14-A, were monitored in the study. For comparison, themetabolic conversion of famciclovir to penciclovir was also assessed.

Hepatocytes Suspension

Cryopreserved hepatocytes plating medium (Cat. #: PY-HMD-01) waspurchased from RILD Research Institute for Liver Diseases (Shanghai) Co.Ltd. Cryopreserved human hepatocyte (Cat. #: X008005, Lot #:VRR) waspurchased from In Vitro Technologies (Baltimore, Md.).

The stock hepatocyte suspension was prepared from cryopreservedhepatocytes in plating medium at the concentration of 1.8×10⁶ cells/mL.

Working Solutions of Compounds

Compounds were dissolved in DMSO to make 50 mM stock solutions. 10 μL ofthe stock solution was diluted to 5 mL plating medium to get a 100 μMworking solution.

Incubations

Reaction suspensions were prepared in 24-well cell culture plate bymixing 200 μL of hepatocytes suspension (Cyno or human) and 200 μL ofworking solution. The final incubation contained 0.9×10⁶ cells/mL and 50μM compound. The above mixtures were incubated at 37° C. in a humidified5% CO₂ atmosphere, with a 150 rpm shaking.

Preparation of Samples for Analysis

After 180 min of incubation, 200 μL of the incubation mixture wastransferred to 1.5 mL tube and quenched with 400 μL stop solution(ice-cold acetonitrile with 0.2 μM Tolbutamide as internal standard).The samples were centrifuged at 12000 rpm for 10 minutes and theresultant supernatants were subjected to LC-MS/MS analysis.

The calibration curves were prepared in the following way. To a 200 μLof cell suspension (cell density of 1.8 million cells/mL), 198 μL ofhepatocyte plating medium and 2 μL of the appropriate concentration ofthe compound in DMSO were added. Samples were mixed thoroughly and 200μL of the mixture was transferred to 400 uL of the stop solution (seeabove). The standard curve range is from 1 μM to 25 μM.

Bioanalysis

The compounds were quantified on an API5500 LC-MC/MC instrument in theESI-Positive MRM mode. The results of prodrug conversion and metabolitegeneration are summarized in Table 2.

TABLE 2 Concentration of the metabolites formed in human hepatocytesafter 3-hour incubation of 50 μM of prodrugs. Metabolized ProductConcentration Example No. Product in human hepatocytes (μM)  2-A  1-A8.7  3-A  1-A 19.3  4-A  1-A 10.3 16-A 14-A 7.3 17-A 14-A 7.3Famciclovir Penciclovir 23.5

In human hepatocytes, compounds of Example 2-A, Example 3-A, Example4-A, Example 16-A and Example 17-A as well as famciclovir weremetabolized to yield the corresponding active metabolites of Example1-A, Example 14-A and penciclovir, respectively.

Example 45 TLR7 Agonist Example 1-AAactivates Murine TLR7

The potency of the TLR7 agonist Example 1-A activating murine TLR7 wasassessed using a stable HEK293-Blue-mTLR7 cell line available fromInvivoGen (Cat. #: hkb-mt1r7, San Diego, Calif., USA). Similar to theHEK293-Blue-hTLR7 as described in Example 43, the HEK293-Blue-mTLR7 isdesigned for studying the stimulation of murine TLR7 by monitoring theactivation of NF-κB. A SEAP reporter gene was placed under the controlof the IFN-β minimal promoter fused to five NF-κB and AP-1-bindingsites. The SEAP expression was induced by the activation of NF-κB andAP-1 upon stimulation of murine TLR7 with TLR7 ligands. SEAP expressionin cell culture supernatant was determined using a QUANTI-Blue™ kit(Cat. #: rep-qb1, Invivogen, San Diego, Calif., USA), a detection mediumthat turns purple/blue in the presence of alkaline phosphatase, at awavelength of 655 nm.

HEK293-Blue-mTLR7 cells were incubated at a density of 250,000˜450,000cells/mL in a volume of 180 μL in a 96-well plate in Dulbecco's ModifiedEagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v)heat-inactivated fetal bovine serum for 24 hours. The HEK293-cells werethen incubated with 20 μL of test compound in a serial dilution in thepresence of final 1% DMSO at 37° C. in a CO₂ incubator for 20 hours. 20μL of the supernatant from each well was incubated with 180 μLQuanti-blue substrate solution at 37° C. for 2 hours and the absorbancewas measured at 655 nm using a spectrophotometer.

As shown in FIG. 1, Example 1-A activates murine TLR7 in a dosedependent manner with an EC50 of 71.8 μM.

Example 46 TLR7 Agonist Example 1-A Induces Antiviral Cytokines inMurine Peripheral Blood Mononuclear Cells (PBMC) In Vitro

To demonstrate TLR7 activation on leukocytes by the compound of thisinvention, murine PBMC (available from ALLCells, LLC.) were subjected tostimulation by Example 1-A. 70 million murine PBMC (C57b1/6 strain) wereseeded into each well of a 24-well plate at the concentration of2×10⁶/mL in 1.5 mL RPMI-1640 medium containing 10% fetal bovine serum.The seeded PBMC were incubated with Example 1-A over a concentrationrange from 2 to 10 μM for 24 hours. 50 μL of cell culture medium wascollected and analyzed with a 36plex Procarta multiplex kit (ebioscienceEPX360-26092-901, eBioscience), which measured the levels of 15 cytokineanalytes, following the manufacturer's instruction.

As shown in FIG. 2, high levels of IFNα, IP10, TNFα, and IL-6 were foundinduced by Example 1-A in a dose-dependent manner. The increased levelsof these cytokines in the stimulated PBMC demonstrate that TLR7 agonistExample 1-A induces immune activation and has the potential to treatinfectious diseases.

Example 47 Example 4-A Reduces HBV DNA and HBsAg in AAV-HBV Model

Example 4-A was evaluated for its in vivo antiviral efficacy using anAAV-HBV mouse model. This mouse model for HBV infection was generated byinjecting C57BL/6 mice with a recombinant adeno-associated virus (AAV)carrying a replicable HBV (hepatitis B virus) genome (AAV-HBV). In 2-3weeks post infection, high levels of HBV viral markers, such as HBVgenomic DNA and HBsAg (HBV surface antigen), was detected in the sera ofinfected mice. With persistent HBV viremia and fully competent immunesystem, the AAV-HBV model is suitable for investigating the in vivoefficacy of Example 4-A.

Two independent in vivo studies were conducted to assess the antiviralpotency of Example 4-A at 100 mg/kg and 300 mg/kg respectively. For eachstudy, ten 4-5 week old male C57BL/6 mice, specific pathogen free, wereavailable from Shanghai Laboratory Animal Center of Chinese Academy ofSciences (SLAC) and housed in an animal care facility in individuallyventilated cages under controlled temperature and light conditionsfollowing the Institutional Animal Care guidelines. AAV-HBV virus stockwas purchased from Beijing FivePlus Molecular Medicine Institute(Beijing, China). C57BL/6 mice were injected with 200 μL of recombinantvirus in saline buffer through tail vein injection. The mice were bledon day 14 post injection to monitor the levels of HBsAg, HBeAg, and HBVgenomic DNA in serum, and randomly grouped based on these HBV biomarkerlevels. The grouped mice were then treated following the study design asshown in Table 3.

TABLE 3 In vivo study in AAV-HBV mouse model Treatment Dose Drug Study#Group # Mice# Compound (mg/kg) delivery 1 1 5 Vehicle 0 PO, QOD, 42D 2 5Example 4-A 100 2 3 5 Vehicle 0 4 5 Example 4-A 300

Mice in groups 1 and 3 were treated with vehicle placebo (2% Klucel LF,0.1% Polysorbate 80, and 0.1% Parabens in water); Mice in groups 2 and 4were orally dosed with Example 4-A at 100 mg/kg and 300 mg/kgrespectively, every other day (QOD). All the mice were treated for atotal of 6 weeks. Serum samples were collected twice a week to monitorthe levels of HBV biomarkers. Serum HBsAg was measured using CLIA kits(Autobio Diagnostics Co., Ltd, Zhengzhou, China) according to themanufacturer's instructions. The lower limit of quantification (LLQ) forHBsAg was 0.1 ng/mL. Serum dilution of 500-fold (for HBsAg) was used toobtain values within the linear range of the standard curve. Serum HBVDNA was extracted using a MagNA Pure 96 DNA and Viral NA Small VolumeKit (Roche) following the manufacturer's instructions. The DNA sampleswere analyzed by real-time quantitative PCR (qPCR) using a HBV-specificprimer and probe set for specific amplification and detection of a 128bp HBV genome region from the nucleotide 2969 to 3096. The LLQ for HBVDNA was 20 copies/μL.

As shown in FIG. 3, after the 6-week treatment, Example 4-A at 100 mg/kginduced more than 2-log reduction in HBV DNA and 1.5-log reduction inHBsAg. At a higher dose as 300 mg/kg, Example 4-A reduced HBV DNA bymore than 3-log and HBsAg by 2.7-log at the end of the treatment. Theresults of this study clearly demonstrate the in vivo antiviral efficacyof Example 4-A and underscore the potential of compounds of thisinvention to develop novel therapy for infectious diseases.

1.-36. (canceled)
 37. A substantially diastereomerically pure compoundof formula (Ia),

wherein R¹ is hydroxy or C₁₋₆alkylcarbonyl-O—; R² is C₁₋₆ alkyl; and R³is hydrogen or C₁₋₆alkylcarbonyl; or a pharmaceutically acceptable saltthereof.
 38. The substantially diastereomerically pure compoundaccording to claim 36, wherein R¹ is hydroxy or acetyloxy; R² is ethylor propyl; and R³ is hydrogen, acetyl or isobutyryl; or apharmaceutically acceptable salt, enantiomer or diastereomer thereof.39. A compound that is5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1R)-1-hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione,having the following structure:

or a pharmaceutically acceptable salt thereof.
 40. A compound that is5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1S)-1-hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione, having the following structure:

or a pharmaceutically acceptable salt thereof.
 41. A compound that is[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-[(1R)-1-hydroxypropyl]tetrahydrofuran-3-yl]acetate, having the following structure:

or a pharmaceutically acceptable salt thereof.
 42. A compound that is[(2R,3R,5S)-2-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-5-[(1S)-1-hydroxypropyl]tetrahydrofuran-3-yl]acetate, having the following structure:

or a pharmaceutically acceptable salt thereof.
 43. A compound that is[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]acetate,having the following structure:

or a pharmaceutically acceptable salt thereof.
 44. A compound that is5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1R)-1-hydroxybutyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione,having the following structure:

or a pharmaceutically acceptable salt thereof.
 45. A compound that is5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1S)-1-hydroxybutyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione,having the following structure:

or a pharmaceutically acceptable salt thereof.
 46. A compound that is[(1S)-1-[(2S,4R,5R)-5-(5-amino-2,7-dioxo-6H-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]2-methylpropanoate, having the following structure:

or a pharmaceutically acceptable salt thereof.
 47. A substantiallydiastereomerically pure compound of formula (IIa),

wherein R⁴ is hydroxy or C₁₋₆alkylcarbonyl-O—; R⁵ is C₁₋₆alkyl orC₃₋₇cycloalkyl; and R⁶ is hydrogen or C₁₋₆alkylcarbonyl; or apharmaceutically acceptable salt, enantiomer or diastereomer thereof.48. The substantially diastereomerically pure compound according toclaim 47, wherein R⁴ is hydroxy or acetyloxy; R⁵ is ethyl orcyclopropyl; and R⁶ is hydrogen or acetyl; or a pharmaceuticallyacceptable salt, enantiomer or diastereomer thereof.
 49. A compound thatis5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1R)-1-hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one,having the following structure:

or a pharmaceutically acceptable salt thereof.
 50. A compound that is5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(1S)-1-hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one,having the following structure:

or a pharmaceutically acceptable salt thereof.
 51. A compound that is[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-[(1R)-1-hydroxypropyl]tetrahydrofuran-3-yl]acetate, having the following structure:

or a pharmaceutically acceptable salt thereof.
 52. A compound that is[(2R,3R,5S)-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-5-[(1S)-1-hydroxypropyl]tetrahydrofuran-3-yl]acetate, having the following structure:

or a pharmaceutically acceptable salt thereof.
 53. A compound that is[(1R)-1-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl]acetate, having the following structure:

or a pharmaceutically acceptable salt thereof.
 54. A compound that is[(1S)-1-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate,having the following structure:

or a pharmaceutically acceptable salt thereof.
 55. A pharmaceuticalcomposition comprising a compound according to any one of claims 37 to54, or a pharmaceutically acceptable salt thereof, and a therapeuticallyinert carrier.
 56. A method for treating a Hepatitis B virus infectioncomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound according to any one of claims 37 to 54,or a pharmaceutically acceptable salt thereof.
 57. A method ofactivating the TLR7 receptor in a patient in need thereof comprisingadministering an effective amount of a compound according to any one ofclaims 37 to 54, or a pharmaceutically acceptable salt thereof.
 58. Amethod of stimulating production of interferon-a comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound any one of claims 37 to 54, or a pharmaceuticallyacceptable salt thereof.